Split inteins with exceptional splicing activity
11142550 · 2021-10-12
Assignee
Inventors
Cpc classification
C07K16/2851
CHEMISTRY; METALLURGY
C07K2319/92
CHEMISTRY; METALLURGY
A61K47/6803
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
C12N9/50
CHEMISTRY; METALLURGY
International classification
C07K14/00
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
Abstract
Embodiments of the present invention relate to inteins, split inteins, compositions comprising inteins and methods for use of these.
Claims
1. A split intein N-fragment comprising an amino acid sequence of at least 98% sequence identity to TABLE-US-00038 (SEQ ID NO: 1) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGL or to (SEQ ID NO: 2) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGL P.
2. A complex comprising the split intein N-fragment of claim 1 and a compound.
3. The complex of claim 2, wherein the compound is selected from the group consisting of (i) a peptide or a polypeptide, (ii) an antibody chain, (iii) an antibody heavy chain and (iv) a compound comprising a peptide, an oligonucleotide, a drug or a cytotoxic molecule.
4. A split intein C-fragment comprising an amino acid sequence of at least 98% sequence identity to TABLE-US-00039 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, to (SEQ ID NO: 4) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN or to (SEQ ID NO: 389) VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.
5. A complex comprising the split intein C-fragment of claim 4 and a compound.
6. The complex of claim 5, wherein the compound is selected from the group consisting of: (i) a peptide or a polypeptide, (ii) a compound comprising a peptide, an oligonucleotide, a drug, or a cytotoxic molecule, (iii) a 1,2-amino thiol bonded to a peptide, an oligonucleotide, a drug, or a cytotoxic molecule, (iv) a 1,2-amino alcohol bonded to a peptide, an oligonucleotide, a drug, or a cytotoxic molecule, and (v) a dendrimer.
7. The complex of claim 5, wherein the compound is a dendrimer having the structure ##STR00012## wherein R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen (H) and cargo molecules.
8. The complex of claim 7, wherein R1, R2, R3, and R4 are each a dye molecule or wherein R1, R2, R3, and R4 are each a fluorescein derivative having the structure ##STR00013##
9. A complex selected from the group consisting of: (i) a complex of the structure ##STR00014## wherein IntC is the split intein C-fragment of claim 4, and wherein n is from 0 to 8, (ii) a complex of the structure ##STR00015## wherein IntC is the split intein C-fragment of claim 4, and wherein n is from 0 to 8, and (iii) a complex of the structure ##STR00016## wherein IntC is the split intein C-fragment of claim 4, and wherein X is sulfur (S) or oxygen (O).
10. A composition comprising: the split intein N-fragment of claim 1; and a split intein C-fragment split intein C-fragment comprising: an amino acid sequence of at least 98% sequence identity to TABLE-US-00040 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, (SEQ ID NO: 4) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, or (SEQ ID NO: 389) VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.
11. A nucleotide plasmid comprising a nucleotide sequence encoding the split intein N-fragment of claim 1.
12. A nucleotide plasmid comprising a nucleotide sequence encoding the split intein C-fragment of claim 4.
13. A method for splicing two complexes comprising: contacting a first complex comprising a first compound and the split intein N-fragment of claim 1 and a second complex comprising a second compound and a split intein C-fragment, wherein the split intein C-fragment comprises an amino acid sequence of at least 98% sequence identity to TABLE-US-00041 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, (SEQ ID NO: 4) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, or (SEQ ID NO: 389) VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN, wherein the contacting is performed under conditions that permit binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and reacting the intein intermediate to form a conjugate of the first compound with the second compound.
14. A method selected from the group consisting of: (i) a method comprising: contacting a first complex comprising a first compound and the split intein N-fragment of claim 1 and a second complex comprising a second compound and a split intein C-fragment, wherein the contacting is performed under conditions that permit binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and reacting the intein intermediate with a nucleophile to form a conjugate of the first compound with the nucleophile; and (ii) a method comprising: fusing a first nucleotide sequence encoding an amino acid sequence of the split intein N-fragment of claim 1 with a second nucleotide sequence encoding an amino acid sequence of a split intein C-fragment, so that the fusion of the first nucleotide sequence and the second nucleotide sequence encodes for a contiguous intein; wherein the split intein C-fragment comprises an amino acid sequence of at least 98%, sequence identity to TABLE-US-00042 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, (SEQ ID NO: 4) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, or (SEQ ID NO: 389) VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.
15. The method of claim 14, wherein; the first compound is a polypeptide or an antibody, or the second compound is a dendrimer or a polypeptide.
16. An intein comprises an amino acid sequence of at least 98% sequence identity to TABLE-US-00043 (SEQ ID NO: 390) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRG EQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLPV KIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.
17. A kit for splicing two complexes together comprising: the split intein N-fragment of claim 1; a split intein C-fragment comprises an amino acid sequence of at least 98% sequence identity to TABLE-US-00044 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, (SEQ ID NO: 4) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, or (SEQ ID NO: 389) VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN; a reagent for binding the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and a nucleophilic agent.
18. A gene fusion comprising: a first nucleotide sequence encoding an amino acid sequence of the split intein N-fragment of claim 1; fused with a second nucleotide sequence encoding a split intein C-fragment comprising an amino acid sequence of at least 98% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3), MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 4), or VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN (SEQ ID NO: 389).
19. The gene fusion of claim 18, comprising: a first nucleotide sequence encoding the amino acid sequence of a split intein N-fragment comprising TABLE-US-00045 (SEQ ID NO: 1) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRG EQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGL, fused with a second nucleotide sequence encoding an amino acid sequence of a split intein C-fragment comprising TABLE-US-00046 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.
20. A polynucleotide encoding the split intein N-fragment of claim 1.
21. A polynucleotide encoding the split intein C-fragment of claim 4.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(2) NpuN corresponds to SEQ ID NO: 5,
(3) NpuC corresponds to SEQ ID NO: 6,
(4) CfaN corresponds to SEQ ID NO: 2, and
(5) CfaC corresponds to SEQ ID NO: 3;
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(16) FIG. 7A3 corresponds to the last two amino acids of the sequences of
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DETAILED DESCRIPTION
(35) Embodiments of the invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent parts can be employed and other methods developed without parting from the spirit and scope of the invention. All references cited herein are hereby incorporated by reference in their entirety as if each had been individually incorporated.
(36) Embodiments of the invention include a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
(37) TABLE-US-00018 (SEQ ID NO: 1) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGL.
(38) Embodiments of the invention include a split intein N-fragment comprising an amino acid sequence, wherein said amino acid sequence comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
(39) TABLE-US-00019 (SEQ ID NO: 2) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGL P.
(40) Embodiments of the invention include a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
(41) TABLE-US-00020 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.
(42) Embodiments of the invention include a split intein C-fragment comprising an amino acid sequence, wherein said amino acid sequence of said C-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
(43) TABLE-US-00021 (SEQ ID NO: 4) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.
(44) Embodiments of the invention include a composition comprising the following: a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1); and a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3).
(45) Embodiments of the invention include a nucleotide plasmid comprising a nucleotide sequence encoding for a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
(46) TABLE-US-00022 (SEQ ID NO: 1) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHN RGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGL.
(47) Embodiments of the invention include a nucleotide plasmid comprising a nucleotide sequence encoding for a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
(48) TABLE-US-00023 (SEQ ID NO: 3) VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.
(49) Embodiments of the invention include a method for splicing two complexes comprising: contacting a first complex comprising a first compound and a split intein N-fragment and a second complex comprising a second compound and a split intein C-fragment, wherein contacting is performed under conditions that permit binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and reacting the intein intermediate to form a conjugate of the first compound with the second compound, wherein said split intein N-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1), and wherein said split intein C-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3). In some embodiments, reacting the intein intermediate comprises contacting the intein intermediate with a nucleophile. In some embodiments, said first compound is a polypeptide. In some embodiments, said first compound is an antibody.
(50) Embodiments of the invention include an intein comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
(51) TABLE-US-00024 (SEQ ID NO: 390) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR GEQEVFENCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGL PVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.
(52) Embodiments of the invention include a kit for splicing two complexes together comprising the following: a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1); a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3); reagents for permitting the binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and a nucleophilic agent.
(53) Embodiments of the invention include a method for generating a synthetic consensus intein peptide sequence comprising: generating a population of a plurality of homologous intein peptide sequences; identifying amino acids associated with fast splicing within said population of a plurality of homologous intein peptide sequences; generating a subpopulation of a second plurality of homologous intein peptide sequences, wherein said second plurality of homologous intein peptide sequences comprise amino acids associated with fast splicing; creating an alignment of at least three peptide sequences of said subpopulation; determining a most frequently occurring amino acid residue at each position of said at least three peptide sequences; and generating a synthetic consensus intein peptide sequence based on said most frequently occurring amino acid residue at each position of said at least three peptide sequences.
(54) Embodiments of the invention include a method comprising: fusing a first nucleotide sequence encoding an amino acid sequence of a first intein fragment comprising CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1) with a second nucleotide sequence encoding an amino acid sequence of a second intein fragment comprising VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3), so that the fusion of the first nucleotide sequence and second nucleotide sequence codes for a contiguous intein.
(55) Embodiments of the invention include a gene fusion comprising a first nucleotide sequence encoding an amino acid sequence of a first intein fragment comprising CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1) fused with a second nucleotide sequence encoding an amino acid sequence of a second intein fragment comprising VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3).
(56) Embodiments of the invention include a contiguous intein that can be used, for example, in traditional semi-synthesis applications such as Expressed Protein ligation.
(57) In some embodiments, the various intein fragments described are linked, fused, chemically bonded, complexed or coupled by conventional methods known in the art to polymers, peptides, polypeptides, oligopeptides, small molecules, nucleotides, polynucleotides, oligonucleotides, drugs, cytotoxic molecules or combinations thereof.
Example 1
(58) In some embodiments, the basis of rapid protein splicing through a comparative study of the first two characterized split inteins, Npu and Ssp was investigated. The substantial difference in splicing rate between these two proteins is especially puzzling given their highly similar sequences (63% identity) and near superimposable active site structures. Previous mutagenesis studies on Npu and Ssp suggest that the difference in activity between the two is likely due to the combined effects of several residues, rather than a single site..sup.6,8 However, it remains unclear just how many residues are responsible for the fast versus slow reaction rates and by extension, whether these ‘accelerator’ residues contribute equally to the individual chemical steps in the overall protein splicing process. Consequently, we began our study by exploring these questions, in the hope that this would provide a starting point for developing an improved PTS system.
(59) The high level of conservation within the active sites of Npu and Ssp suggests that more distal amino acid differences account for the disparity in splicing rate between the two. Thus, attention was focused on ‘second shell’ residues, those directly adjacent to the active site. To simplify this analysis, a batch mutagenesis strategy was employed in conjunction with a previously reported in vitro PTS assay..sup.5 This assay uses split intein constructs with short native extein sequences and allows the rates of branched intermediate formation (k.sub.1,k.sub.2) and its resolution to final splice products (k.sub.3) to be determined using a three state kinetic model.
(60) The known cross-reactivity of Npu and Ssp intein fragments served as a convenient platform on which to assess which half of the split intein contributes most significantly to the difference in activity..sup.3 Both the Ssp.sup.N-Npu.sup.C (chimera 1) and Npu.sup.N-Ssp.sup.C (chimera 2) chimeras show a decrease in the rates of branch formation and resolution compared to that of native Npu (
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(62) Next the individual contributions of residues within batch mutants 1 and 2 was investigated, since these had the most profound effect on splicing activity. For Batch 2, further mutagenesis shows that the interaction between F56, K70, and D85 is likely responsible for the increased rate of branch formation in Npu.sup.N (
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(65) The ‘accelerator’ residues found to affect the splicing rate allow for an activity-guided approach to engineer a consensus DnaE intein. Consensus protein engineering is a tool applied to a homologous set of proteins in order to create a thermostable variant derived from the parent family..sup.13,14 A multiple sequence alignment (MSA) is first generated from homologues of a particular protein, from which the most statistically frequent residue at each position is chosen as the representative in the consensus sequence. For the DnaE inteins, 105 sequences were identified through a BLAST.sup.15 search of the JGI.sup.16 and NCBI.sup.17 databases (
(66) TABLE-US-00025 >NpuPCC73102/1-137 (SEQ ID NO: 5) CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNL PN (SEQ ID NO: 6) IKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN >CthPCC7203: /1-137 Chroococcidiopsis thermalis PCC 7203 (SEQ ID NO: 7) CLSYDTEILTVEYGAIPIGKIVEERIECTVYSVDNNGFIYTQPIAQWHNR GQQEVFEYCLEDGSIIRATKDHKFMTFEGKMLPIDEIFEQELDLKQVKSI QN (SEQ ID NO: 8) VKIISRKSLGIQPVYDIGVERDHKFVLKNGLVASN >NspCCY9414: /1-137 Nodularia spumigena CCY9414 genome (SEQ ID NO: 9) CLSYDTEILTVEYGYIPIGEIVEKAIECSVYSVDNNGNVYTQPIAQWHNR GEQEVFEYSLEDGSTIRATKDHKFMTTDGQMLPIDEIFAQELDLLQVHGL PK (SEQ ID NO: 10) VKITARKFVGRENVYDIGVERYHNFAIKNGLIASN >AcyPCC7122: /1-137 Anabaena cylindrica PCC 7122 (SEQ ID NO: 11) CLSYDTEVLTVEYGFIPIGEIVEKRIECSIFSVDKNGNVYTQPIAQWHNR GRQEIYEYCLDDGSKIRATKDHKFMTTAGEMLPIDEIFERDLDLLKVEGL PE (SEQ ID NO: 12) VKIISRQYLGQADVYDIGVEEDHNFAIKNGFIASN >CspPCC7507: /1-137 Calothrix sp. PCC 7507, complete genome (SEQ ID NO: 13) CLSYDTEVLTVEYGLLPIGEIVEKGIECRVFSVDNHGNVYTQPIAQWHNR GQQEVFEYGLDDGSVIRATKDHKFMTTDGKMLPIDEIFERGLDLLQVQGL PE (SEQ ID NO: 14) VKVITRKYIGKENVYDIGVELDHNFAIRNGLVASN >NspPCC7524: /1-137 Nostoc sp. PCC 7524 (SEQ ID NO: 15) CLSYDTEILTVEYGFLPIGEIVEKGIECTVFSVASNGIVYTQPIAQWHNR GQQEIFEYCLEDGSIIRATKDHKFMTQDGQMLPIDEIFACELDLLQVQGL PE (SEQ ID NO: 16) VKVVTRKYIGKENVYDIGVERDHNFVIRNGLVASN >Nax0708: /1-137 ‘Nostoc azollae’ 0708 (SEQ ID NO: 17) CLSYKTEVLTVEYGLIPIGEIVEKRIECSLFSVDENGNIYTQPIAQWHHR GVQEVYEYCLDDGTIIRATKDHKFMTTIGEMLPIDEIFERDLNLLQVNGL PT (SEQ ID NO: 18) VKIISRQFLGPANVYDIGVAQDHNFAIKNGLIASN >NspPCC7120: /1-137 Nostoc sp. PCC 7120 DNA (SEQ ID NO: 19) VLSYDTEVLTVEYGFVPIGEIVEKGIECSVFSINNNGIVYTQPIAQWHHR GKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGL PE (SEQ ID NO: 20) IKIASRKFLGVENYDIGVRRDHNFFIKNGLIASN >AvaATCC29413/ 1-137 Anabaena variabilis ATCC 29413 (SEQ ID NO: 21) VLSYDTEVLTVEYGFVPIGEIVDKGIECSVFSIDSNGIVYTQPIAQWHHR GKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLGVKGL PE (SEQ ID NO: 22) IKIASRKFLGVENVYDIGVGRDHNFFVKNGLIASN >PspPCC7327: /1-135 Pleurocapsa sp. PCC 7327. (SEQ ID NO: 23) CLSYDTKILTVEYGAMPIGKIVEEQIDCTVYTVNQNGFVYTQPIAQWHDR GKQEIFEYCLEDGSIIRATKDHKFMTTDGQMLPIDKIFEKGLDLKTINCD (SEQ ID NO: 24) VKILSRKSLGIQSVYDIGVEKDHNFLLANGLVASN >CspPCC7424: /1-135 Cyanothece sp. PCC 7424 (SEQ ID NO: 25) CLSYETQIMTVEYGLMPIGKIVEEQIDCTVYTVNKNGFVYTQPIAQWHYR GEQEVFEYCLEDGSTIRATKDHKFMTTDGQMLPIDEIFEQGLELKQIHLS (SEQ ID NO: 26) VKIISRQSLGIQPVYDIGVEKDHNFLISDGLIASN >CspPCC7822: /1-134 Cyanothece sp. PCC 7822 (SEQ ID NO: 27) CLSYDETEILTVEYGPMPIGKIVEEQIECTVYTVDKNGLVYTQPIAQWHH RGQQEVFEYCLEDGSIIRATKDHKFMTDDGQMLPIEEIFEKGLELKQIIL (SEQ ID NO: 28) VKIISRQLAGNQTVYDLGVEKDHNFLLANGLIASN >NspPCC7107: /1-137 Nostoc sp. PCC 7107 (SEQ ID NO: 29) VLSYDTQVLTVEYGLVPIGEIVEKQLECSVFTIDGHGYVYTQAIAQWHNR GQQEVFEYGLEDGSVIRATKDHKFMTTDGQMLPIDEIFERELDLLQVQGL RW (SEQ ID NO: 30) VKIITRKYIGQANVYDIGVAQDHNFVIENRLIASN >TboIicb1/1-136 Tolypothrix bouteillei Iicb1 (SEQ ID NO: 31) VLSYDTEILTVEYGFLPIGKIVEKGIECNVYSVDKNGNIYTQPIAQWHDR GEQEVFEYCLENGSVIRATKDHKFMTTSGEMLPIDEIFERGLDLIRVEDL P (SEQ ID NO: 32) VKILTRKSIGKQTVYDIGVERDHNFVIKNGSVASN >Aov: /1-136 Aphanizomenon ovalisporum DnaE precursor (dnaE) gene (SEQ ID NO: 33) CLSADTEILTVEYGFLPIGEIVGKAIECRVYSVDGNGNIYTQSIAQWHNR GEQEVFEYTLEDGSIIRATKDHKFMTTDGEMLPIDEXFARQLDLMQVQGL H (SEQ ID NO: 34) VKITARKFVGRENVYDIGVEHHHNFAIKNGLIASN >OnvPCC7112: /1-137 Oscillatoria nigro-viridis PCC 7112 (SEQ ID NO: 35) CLSYDTKILTVEYGPMAIGKIVEEKIECTVYSVDSNGYIYTQSIAQWHRR GQQEVFEYCLEDGSIIRATKDHKFMTVGGQMLPIDEIFEQGLDLKQINSS SD (SEQ ID NO: 36) VKIISRKSLGTQEVYDIGVEREHNFILENSLVASN >RspPCC7116: /1-135 Rivularia sp. PCC 7116, complete genome (SEQ ID NO: 37) CLSYDTEVLTEEFGLIPIGKIVEEKIDCTVYSVDVNGNVYSQPIAQWHNR GMQEVFEYELEDGSTIRATKDHKFMTVDGEMLAIDEIFEKGLELKRVGIY (SEQ ID NO: 38) VKIISRKVLKTENVYDIGLEGDHNFIIKDGLIASN >TerIMS101: /1-137 Trichodesmium erythraeum IMS101 (SEQ ID NO: 39) CLTYETEIMTVEYGPLPIGKIVEYRIECTVYTVDKGYIYTQPIAQWHNRG MQEVYEYSLEDGTVIRATPEHKFMTEDGQMLPIDEIFERNLDLKCLGTLE L (SEQ ID NO: 40) VKIVSRKLAKTENVYDIGVTKDHNFVLANGLIASN >MspPCC7113: /1-137 Microcoleus sp. PCC 7113, (SEQ ID NO: 41) CLSYDSEILTVEYGLMPIGKIVEEGIECTVYSVDSHGYLYTQPIAQWHHR GQQEVFEYDLEDGSVIRATKDHKFMTSEGQMLAIDEIFERGLELKQVKRS QP (SEQ ID NO: 42) VKIVRRKSLGIQTVYDIGVERDHNFLLANGLVASN >ScyPCC7437: /1-137 Stanieria cyanosphaera PCC 7437 (SEQ ID NO: 43) CLSYDTEILTVEYGAMPIGKIVKEQIECNVYTVNQNGFIYPQAIAQWHER GKQEIFEYTLDNGLVIRATKDHKFMTIDGQMLPIDEIFERGLELQRINDY SN (SEQ ID NO: 44) VKIVSRKSLGKQPVYDIVTKDHNFLLSNGVVASN >CspPCC6303: 1-137 Calothrix sp. PCC 6303 (SEQ ID NO: 45) CLSYDTEILTWEYGFLKIGEIVEKQILCSVFSVDEQGNVYTQPIAQWHNR GLQELFAYQLEDGGVIRATKDHKFMTTDGQMLAIDEIFERQLDLFQVKGL PE (SEQ ID NO: 46) VKIISRKVLKTENVYDIGLEGDHNFIIKDGLIASN >Cst/ 1-134 PCC7202: Cyanobacterium stanieri PCC 7202 (SEQ ID NO: 47) CLSYDTEVLTVEYGVLPIGKIVEEQIQCTVYSVDQYGFVYTQAIAQWHDR GEQEVFEYELENGATIKATKDHKMMTSDGQMLPIDQIFEQGLDLFMVSF (SEQ ID NO: 48) VKIVKRRSHGIQKVYDIGVAKDHNFLLHNGLVASN >CspATCC51142: /1-134 Cyanothece sp. ATCC 51142 (SEQ ID NO: 49) CLSYDTEILTVEYGPMPIGKIVEENINCTVYTVDPNGFVYTQAIAQWHYR GEQEIFEYYLEDGATIRATKDHKFMTMEGKMLPIDEIFENNLDLKQLTL (SEQ ID NO: 50) VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN >CspPCC8801: /1-134 Cyanothece sp. PCC 8801 (SEQ ID NO: 51) CLSYDTEILTVEYGAIPIGKVVEENIDCTVYTVDKNGFVYTQNIAQWHLR GQQEVFEYYLDDGSILRATKDHQFMTLEGEMLPIHEIFERGLELKKIKI (SEQ ID NO: 52) VKIVSYRSLGKQFVYDIGVAQDHNFLLANGSIASN >Asp: /1-136 Anabaena sp. 90 chromosome (SEQ ID NO: 53) CLSYDTEILTVEYGFLEIGEIVEKQIECKVYTIDSNGMLYTQSIAQWHNR GQQEVYEYLLENGAIIRATKDHKFMTEAGQMLPIDEIFAQGLDLLQVGVA E (SEQ ID NO: 54) VKIVSRTYVGQANVYDIGVESDHNFVIKNGFIASN >Aha: /1-137 Aphanothece halophytica (SEQ ID NO: 55) CLSYDTEIWTVEYGAMPIGKIVEEKIECSVYTVDENGFVYTQPIAQWHPR GQQEIIEYTLEDGRKIRATKDHKMMTESGEMLPIEEIFQRELDLKVETFH EM (SEQ ID NO: 56) VKIIKRQSLGRQNVYDVCVETDHNFVLANGCVASN >HspPCC7418: /1-137 Halothece sp. PCC 7418 (SEQ ID NO: 57) CLSYDTEIWTVEYGAMPIGKIVEEKIECSVYTVDENGFVYTQPIAQWHPR GQQEIIEYTLEDGRKIRATKDHKMMTESGEMLPIEEIFQRELDLKVETFH EM (SEQ ID NO: 58) VKIIKRQSLGRQNVYDIGVETDHNFVLANGCVASN >CapPCC10605: /1-137 Cyanobacterium aponium PCC 10605 (SEQ ID NO: 59) VLSYDTEILTVEYGAISIGKIVEEKINCQVYSVDKNGFIYTQNIAQWHDR GSQELFEYELEDGRIIKATKDHKMMTKDGQMLAINDIFEQELELYSVDDM GV (SEQ ID NO: 60) VKIVKRRSLGVQPVYDIGVEKDHNFILANGLVASN >Cat: /1-133 Candidatus Atelocyanobacterium thalassa isolate (SEQ ID NO: 61) CLSYDTKVLTVEYGPLPIGKVVQENIRCRVYTTNDQGLIYTQPIAQWHNR GKQEIFEYHLDDKTIIRATKEHQFMTVDHVMMPIDEIFEQGLELKKIK (SEQ ID NO: 62) LKIIRRKSLGMHEVFDIGLEKDHNFVLSNGLIASN >Oli: /1-137 Oscillatoria limnetica ‘Solar Lake’ DnaE precursor (SEQ ID NO: 63) CLSYNTEVLTVEYGPLPIGKIVDEQIHCRVYSVDENGFVYTQAIAQWHDR GYQEIFAYELADGSVIRATKDHQFMTEDGQMFPIDEIWEKGLDLKKLPTV QD (SEQ ID NO: 64) VKIVRRQSLGVQNVYDIGVEKDHNFLLASGEIASN >Cen: /1-137 Cyanobacterium endosymbiont of Epithemia turgida (SEQ ID NO: 65) CLSYDTEVLTVEYGAIPIGRMVEESLDCTVYTVDKNGFVYTQSIQQWHSR GQQEIFEYCFEDGSIIRATKDHKFMTAEGKMSSIHDIFEQLELKKIIPWS G (SEQ ID NO: 66) AKIISCKSLGKQSVYDIGVVQDHNFLLANGVVASN >SspPCC7502: /1-133 Synechococcus sp. PCC 7502 (SEQ ID NO: 67) CLGYDTPVLTVEYGFMPIGKIVEEKIQCHVYSVDQNGLVFTQAIAQWHNR GQQEVWEYNLDNGDIVRATKDHKFMTIDGQMLPINQIFEQGLELKVIA (SEQ ID NO: 68) VKIVSCKPLRVQTVYDIGVEKDHNFILDNGLVASN >DsaPCC8305: /1-134 Dactylococcopsis salina PCC 8305 (SEQ ID NO: 69) CLSYDTEVLTEEYGAIPIGKIVEERMNCHVYSVDENGFIYSQPIAQWHPR GEQEVVEYTLEDGKIIRATADHKMMTETGEMLPIEQIFQQQLDLKISNQ (SEQ ID NO: 70) VKIINRQSLGKQTVYDIGVEKDHNFILGNGLVASN >CstPCC7417: /1-137 Cylindrospermum stagnale PCC 7417 (SEQ ID NO: 71) CLSYDTEILTVEYGFIPIGEIVEKRIECSVYSVDNHGNVYTQPIAQWHNR GLQEVFEYCLEDGSTIRATKDHKFMTTDKEMLPIDEIFERGLDLLRVEGL PI (SEQ ID NO: 72) VKIIMRSYVGRENVYDIGVERDHNFVAKNGLIAAN >SspPCC6803: /1-137 Synechocystis sp. PCC 6803 (SEQ ID NO: 73) CLSFGTEILTVEYGPLPIGKIVSEEINCSVYSVDPEGRVYTQAIAQWHDR GEQEVLEYELEDGSVIRATSDHRFLTTDYQLLAIEEIFARQLDLLTLENI KQ (SEQ ID NO: 74) VKVIGRRLSGVQRIFDIGLPQDHNFLLANGAIAAN >GspPCC7407: /1-137 Geitlerinema sp. PCC 7407 (SEQ ID NO: 75) CLSYETPVMTVEYGPLPIGRIVEEQLDCTVYSVDEQGHVYTQPVAQWHHR GLQEVVEYELEDGRRLRATADHRFMTETGEMLPLAEIFERGLELRQVALR VP (SEQ ID NO: 76) VKIVSRRSLGMQLVYDIGVAADHNFVLADGLIAAN >SspPCC6714: /1-137 Synechocystis sp. PCC 6714 (SEQ ID NO: 77) CLSFDAEILTVEYGPLSIGKIVGEEINCSVYSVDPQGRIYTQAIAQWHDR GVQEVFEYELEDGSVIRATPDHRFLTTDYELLAIEEIFARQMDLLTLTNL KL (SEQ ID NO: 78) VKVVRRRSLGMHRVFDIGLAQDHNFLLANGAIAAN >MaePCC7806: /1-135 Microcystis aeruginosa PCC 7806 (SEQ ID NO: 79) CLGGETLILTEEYGLLPIAKIVSEEVNCTVYSVDKNGFVYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTNDGEMLAIDTIFERGLDLKSSDFS (SEQ ID NO: 80) VKIISRQSLGRKPVYDIGVEKDHNFLLGNGLIASN >MaeNIES843: /1-135 Microcystis aeruginosa NIES-843 DNA (SEQ ID NO: 81) CLGGETLILTEEYGLLPIAKIVSEEINCTVYTVDQNGFVYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTSDGEMLAIDTIFERGLDLKSSDFS (SEQ ID NO: 82) VKIIGRQSLGRKPVYDIGVEKDHNFLLGNGLIASN >AmaMBIC11017: /1-137 Acaryochloris marina MBIC11017, (SEQ ID NO: 83) CLSYDTPVLTLEYGWLPIGQVVQEQIECQVFSINERGHLYTQPIAQWHHR GQQEVFEYTLADGSTIQATAEHQFMTTDGQMYPVQQIFEEGLSLKQLPLP WQ (SEQ ID NO: 84) VKIIQRRSLGLQSVYDIGLAQDHNFVMANGWVAAN >LspPCC7376: /1-137 Leptolyngbya sp. PCC 7376 (SEQ ID NO: 85) CLDGETPIVTVEYGVLPIREIVEKELLCSVYSIDENGFVYTQPVEQWHQR GDRQMFEYQLDNGGVIRATPDHKFLTTEGEMVAIDEIFEKGLNLAEFAPA DL (SEQ ID NO: 86) VKILRRHSIGKAKTYDIGVSKNHNFLLANGLFASN >SelPCC6301: /1-137 Synechococcus elongatus PCC 6301 (SEQ ID NO: 87) CLAADTEVLTVEYGPIAIGKLVEENIRCQVYCCNPDGYIYSQPIGQWHQR GEQEVIEYELSDGRIIRATADHRFMTEEGEMLSLDEIFERSLELKQIPTP LL (SEQ ID NO: 88) VKIVRRRSLGVQPVYDLGVATVHNFVLANGLVASN >SspPCC6312: 1-137 Synechococcus sp. PCC 6312 (SEQ ID NO: 89) CLSADTELYTVEYGWLPIGRLVEEQIECQVLSVNAHGHVYSQPIAQWHRR AWQEVFEYQLETGGTIKATTDHQFLTTDGQMYRIEDIFQRGLDLWQLPPD RF (SEQ ID NO: 90) VKIISRCSLGIQPVYDIGVAQDHNFVIRGGLVASN >Tel: /1-137 Thermosynechococcus elongatus BP-1 DNA (SEQ ID NO: 91) CLSGETAVMTVEYGAVPRIILVQERLSCHVYSLDGQGHLYTQPIAQWHFQ GFRPVYEYQLEDGSTICATPDHRFMTTRGQMLPIEQIFQEGLELWQVAIA PR (SEQ ID NO: 92) GKIVGRRLMGWQAVYDIGLAADHNFVLANGAIAAN >Tsp: /1-137 Thermosynchococcus sp. NK55 genome (SEQ ID NO: 93) CLSGETAVMTVEYGAVPRIILVQERLTCHVYSLDAQGHLYTQPIAQWHFQ GFRPVYEYQLEDGSTIWATPDHRFMTTRGQMLPIEQIFQEGLELWQGPIA PS (SEQ ID NO: 94) CKIVGRQLVGWQAVYDIGVARDHNFLLANGAIAAN >Tvu: /1-137 Thermosynchococcus vulcanus DnaE precursor (SEQ ID NO: 95) CLSGETAVMTVEYGAIPIRRLVQERLICQVYSLDPQGHLYTQPIAQWHFQ GFRPVYAYQLEDGSTICATPDHRFMTTSGQMLPIEQIFREGLELWQVAIA PP (SEQ ID NO: 96) CKIVGRRLVGWQAVYDIGLAGDHNFLLANGAIAAN >SspPCC7002: /1-137 Synechococcus sp. PCC 7002 (SEQ ID NO: 97) CLAGGTPVVTVEYGVLPIQTIVEQELLCHVYSVDAQGLIYAQLIEQWHQR GDRLLYEYELENGQMIRATPDHRFLTTTGELLPIDEIFTQNLDLAAWAVP DS (SEQ ID NO: 98) VKIIRRKFIGHAPTYDIGLSQDHNFLLGQGLIAAN >ShoPCC7110: /1-136 Scytonema hofmanni PCC 7110 contig00136 (SEQ ID NO: 99) CLSYDTEVLTAEYGFLPIGKIVEKAIECTVYSVDNDGNIYTQPIAQWHDR GQQEVFEYSLDDGSVIRATKDHKFMTTGGQMLPIDEIFERGLDLMRIDSL P (SEQ ID NO: 100) VKILTRKSIGKQTVYDIGVERDHNFVIKNGLVASN >WinUHHT291/1-136 Westiella intricata UH HT-29-1 (SEQ ID NO: 101) CLSYDTEILTVEYGFLPIGEIVEKRIECTVYTVDTNGYVYTQAIAQWHNR GEQEVFEYALEDGSIIRATKDHKFMTSEGQMLPIDEIFVKGLDLLQVQGL P (SEQ ID NO: 102) VKIITRKFLGIQNVYDIGVEQNHNFVIKNGLVASN >FspPCC9605: /1-136 Fischerella sp. PCC 9605 FIS9605DRAFT (SEQ ID NO: 103) CLSYDTEILTVEYGFLPIGEIVEKGIECTVYTVDNNGNVYTQTIAQWHNR GQQEVFEYCLEDGSVIRATKDHKFMTTDGQMLPIDEIFARGLDLLQVKNL P (SEQ ID NO: 104) VKIVTRRPLGTQNVYDIGVESDHNFVIKNGLVASN >MrePCC10914: /1-137 Mastigocladopsis repens PCC 10914 (SEQ ID NO: 105) CLSYDTEVLTVEYGFLPIGEIVEKSIECSVYTVDSNGNVYTQPIAQWHNR GQQEVFEYCLEDGSIIRATKDHKFMTIHGQMLPIDEIFERGLELMKIQGL PE (SEQ ID NO: 106) AKIITRKSLGTQNVYDIGVERDHNFVTRDGFIASN >ShoUTEX2349: /1-137 [Scytonema hofmanni] UTEX 2349 (SEQ ID NO: 107) CLSYNSEVLTVEYGFLPIGKIVEKGIECSVYSVDSYGKIYTQVIAQWHNR GQQEVFEYCLEDGTIIQATKDHKFMTVDGQMLPIDEIFERGLDLMQVQGL PD (SEQ ID NO: 108) VKIITRKSLGTQNVYDIGVSSDHNFVMKNGLIASN >AspPCC7108: /1-137 Anabaena sp. PCC 7108 Ana7108scaffold_2_Cont3 (SEQ ID NO: 109) CLSSDTEVLTVEYGLIPIGEIIEKRIDCSVFSVDKNGNIYTQPIAQWHDR GIQELYEYCLDDGSTIRATKDHKFMTTAGEMLPIDEIFERGLDLLKVHNL PQ (SEQ ID NO: 110) VKIITRNYVGKENVYDIGVERDHNFAIKNGLIASN >FspPCC9339: /1-137 Fischerella sp. PCC 9339 PCC9339DRAFT (SEQ ID NO: 111) CLSYDTEVLTVEYGFLPIGEIVEKRIECTVYTVDHNGYVYTQPIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTSEGQMLPIDEIFARELDLLQVTGL VN (SEQ ID NO: 112) VKIVTRRLLGIQNVYDIGVEQNHNFVIKNGLVASN >Csp336: /1-137 Calothrix sp. 336/3 (SEQ ID NO: 113) CLSYDTEIFTVEYGFLPIGEIVEKRLECTVLTVDNHGNIYSQPIAQWHHR QQQIEYEGLEDGSVIRATKDHKFMTTDGQMLPIDEIFERGLDLLQVTNLD N (SEQ ID NO: 114) VKVITRKLADTENVYDIGVENHHNFLIKNGLVASN >FthPCC7521: /1-136 Fischerella thermalis PCC 7521 (SEQ ID NO: 115) CLSYETEILTVEYGFLPIGEIVEKRIECSVYTVDNNGYVCTQPIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTIDRQMLPIDEIFARGLDLLQVTGL P (SEQ ID NO: 116) VKIITRKSLGTQNVYDIGVEQNHNFVIKNGLVASN >CyaPCC77021/1-137 cyanobacterium PCC 7702 Chl7702 (SEQ ID NO: 117) CLSYDTEILTVEYGFLSIGEIVEKEIECTVYTVDSNGYIYTQPIAQWHEQ GEQEIFEYSLEDGSTIRATKDHKFMTIEGEMLPIDQIFARQLDLMQITGL PQ (SEQ ID NO: 118) VKISTKKSLGKQKVYDIGVVRDHNFIIKNGFVASN >FspPCC9431: /1-136 Fischerella sp. PCC 9431 (SEQ ID NO: 119) CLSYDTEVLTVEYGFLPIGEIVEKRIECTVYTVDTNGYVYTQAIAQWHNR DEQEVFEYALEDGSIIRATKDHKFMTSEGQMLPIDEIFAKGLDLLQVQGL P (SEQ ID NO: 120) VKIVTRKFLGIQNVYDIGVEQNHNFVIKNGLVASN >FmuPCC7414: /1-137 Fischerella muscicola PCC 7414 (SEQ ID NO: 121) CLSYETEILTVEYGFLPIGEIVEKRIECSVYTVDNNGYVCTQTIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTIDRQMLPIDEIFARGLDLLQVKGL PE (SEQ ID NO: 122) VKIITRQSLGTQNVYDIGVEQNHNFVIKNGLVASN >FumPCC73103: /1-137 Fischerella muscicola SAG 1427-1 = PCC 73103 (SEQ ID NO: 123) CLSYDTEVLTVEYGFLPIGEIVEKTIECNVFTVDSNGYVYTQPIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTSEGKMLPIDEIFARELDLLQVTGL IN (SEQ ID NO: 124) VKIVTRKFLGIQNVYDIGVEQNHNFVIKNGLVASN >Lae: /1-137 Lnygbya aesgtuarii BL J laest3.contig.3 (SEQ ID NO: 125) CLSYDTEILTVEYGAIPIGKVVDEKIECTVYSVDKNGLIYTQPIAQWHNR GKQEVFEYSLEDGSTIRATKDHKFMTMDNQMLPIDEILEKGLELKQVNAD SV (SEQ ID NO: 126) VKIVSRKSLDSQTVYDIGVETDHNFLLANGSVASN >MspPCC7126: /1-135 Microchaete sp. PCC 7126 (SEQ ID NO: 127) CLSYKTQVLTVEYGLLAIGEIVEKNIECSVFSVDIHGNVYTQPIAWHHRG QQEVFEYGLEDGSIIRATKDHKFMTTQGEMLPIDEIFARGLDLLQVKGV (SEQ ID NO: 128) VKIITRKYIGKENVYDIGVEQDHNFAIKNGLIAAN >Lsp: /1-137 Leptolyngbya sp. JSC-1 (SEQ ID NO: 129) CLSYDTEILTVEYGALPIGKIVENQMICSVYSIDNNGYIYIQPIAQWHNR QQEVFEYILEDGSIIRSTKDHKFMTKGGEMLPIDEIFERGLELAQVTRLE Q (SEQ ID NO: 130) VKIISRRSVGVQSVYDIGVKQDHNFFLRNGLIASN >CwaWH8501: /1-137 Crocosphaera watsonii WH8501 (SEQ ID NO: 131) CLSYDTEILTVEYGAMYIGKIVEENINCTVYTVDKNGFVYTQTIAQWHNR GEQEIFEYDLEDGSKIKATKDHKFMTIDGEMLPIDEIFEKNLDLKQVVSH PD (SEQ ID NO: 132) VKIIGCRSLGTQKVYDIGVEKDHNFLLANGSIASN >CchPCC7420: /1-135 Coleofasciculus chthonplastes PCC 7420 (Mcht) (SEQ ID NO: 133) CLSYDTQILTVEYGAVAIGEIVEKQIECTVYSVDENGYVYTQPIAQWHNR GEQEVFEYLLEDGATIRATKDHKFMTDEDQMLPIDQIFEQGLELKQVEVL (SEQ ID NO: 134) VKIIGRKPLGTQPVYDIGVERDHNFLLFNGSVASN >CspPCC6712/1-133 (SEQ ID NO: 135) CLSYDTEVLTVEYGAIPIGKIVEEKIACNVYSVDKNGFVYTQPIAQYHDR GIQEVFEYRLENGSVIRATKDHKMMTADGQMLPIDEIFKQNLDLKQLN (SEQ ID NO: 136) VKIISRQSLGKQSVFDIGVAKDHNFLLANGLVASN >AflNIES81: /1-132 Aphanizomenon flos-aquae NIES-81 (SEQ ID NO: 137) CLSYDTEILTVEYGFLQIGEIVEKQIECKVYTVDSNGILYTQSIAQWHNR GQQEVYEYLLENGAIIRATKDHKFMTEEGQMLPIDEIFSQGLDLLQV (SEQ ID NO: 138) VKIISRTYVGQANVYDIGVENDHNFVIKNGFIAAN >Rbr: /1-137 Raphidiopsis brookii D9 D9_5, (SEQ ID NO: 139) CLSYETEVLTLEYGFLPIGEIVDKQMVCTVFSVNDSGNVYTQPIGQWHDR GVQELYEYCLDDGSTIRATKDHKFMTTQGEMVPIDEIFHQGWELVQVSGT MN (SEQ ID NO: 140) VKIVSRRYLGKADVYDIGVAKDHNFIIKNGLVASN >CspCCy0110: /1-134 Cyanothece sp. CCY0110 1101676644604 (SEQ ID NO: 141) CLSYDTEILTVEYGPMPIGKIVEENINCSVYTVNKNGFVYTQSIAQWHHR GEQEVFEYYLEDGETIRATKDHKFMTTEGKMLPIDEIFENNLDLKKLTV (SEQ ID NO: 142) VKIIERRSLGKQNVYDIGVEKDHNFLLSNNLIASN >XspPCC7305: /1-135 Xenococcus sp. PCC 7305 (SEQ ID NO: 143) CLSASTEVLTVEYGAISIGKIVEERIECTVYSVDANGFVYTQEIAQWHNR GEQEVFEYMLDDGSVIRATKDHKLMTIDGQMVAIDEIFSQGLELKQVLGL (SEQ ID NO: 144) VKIVSRKSLGTQTVYDLGVARDHNFLLANGTVASN >PspPCC7319: /1-135 Pleurocapsa sp. PCC 7319 (SEQ ID NO: 145) CLSYDTEIYTVEYGALPIGKIVESRIKCTVLTVDKNGLVYSQPIVQWHDR GIQEVFEYTLDNGATIRATKDHKFMTVEGQMLPIDEIFELGLELKEIQQF (SEQ ID NO: 146) VKIISRQSLGKQSVYDIGVAKDHNFLLANGMVASN >CraCS505: /1-137 Cylindrospermopsis raciborskii CS-505 (SEQ ID NO: 147) CLSYETEVLTLEYGFVPIGEIVNKQMVCTVFSLNDSGNVYTQPIGQWHDR GVQGLYEYCLDDGSTIRATKDHKFMTTQGEMVPIDEIFHQGLWELVQVSG ISK (SEQ ID NO: 148) VKIVSRRYLGKADVYDIGVAKDHNFIIKNGLVASN >SmaPCC6313/1-129 Spirulina major PCC 6313 (SEQ ID NO: 149) CLTYDTLVLTVEYGPVPIGKLVEAQINCQVYSVDANGFIYTQAIAQWHDR GQRQVYEYTLEDGSTIRATPDHKFMTATGEMLPIDQIFEQGLDL (SEQ ID NO: 150) VKIIHRRALPPQSVYDIGVERDHNFLLPSGWVASN >SsuPCC9445: /1-131 Spirulina subsalsa PCC 9445 (SEQ ID NO: 151) CLSYDTKIITVEYGAIAIGTIVEQGLHCHVYSVDPNGFIYTQPIAQWHQR QEVFAYTLENGSIIQATKDHKFMTQQGKMLPIDTIFEQGLDLLQ (SEQ ID NO: 152) VKIIKRTSLGVRPVYDIGVIQDHNFLLENGLVASN >MaePCC9807: /1-135 Microcystis aeruginosa 9807 (SEQ ID NO: 153) CLGGETLILTEEYGLLPIAKIVSEEINCTVYSVDKNGFIYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTSDGEMLAIDTIFERGLDLKSSDFS (SEQ ID NO: 154) VKIISRQFLGRKPVYDIGVEKDHNFLLGNGLIASN >MspGI1: /1-130 Myxosarcina sp. GI1 contig_13 (SEQ ID NO: 155) CLSYDTEVLTLKYGALPIGEIVEKRINCHVYTRAESGFFYIQSIEQWHDR GEQEVFEYTLENGATIKATKDHKFMTSGGQMLPIDEIFERGLDLL (SEQ ID NO: 156) VKIVSRKSLGKQPVYDLGVAKDHNFLLANGTVASN >LspPCC6406: /1-136 Leptolyngbya sp. PCC 6406 (SEQ ID NO: 157) CLSADTQLLTVEYGPLEIGRIVEEQIACHVYSVDANGFVYTQPIAQWHSR GEQEIFEYQLEDGRTLRATADHKFMTTTGEMGRINDIFEQGLDLKQIDLP Q (SEQ ID NO: 158) VKVVSRQSLGVQPVYDIGVATDHNFLLADGLVASN >AspCCMEE5410: 1-132 Acaryochloris sp. CCMEE 5410 (SEQ ID NO: 159) CLSYDTPVLTLEYGWLPIGQVVQEQIECQVFSINERGHLYTQPIAQWHHR GQQEVFEYTLTDGSTIQATAEHQFMTTDGQMYPIQQIFEEGLSLKQL (SEQ ID NO: 160) VKITQRRSLGLQSVYDIGLAQDHNFVIANGWVAAN >GhePCC6308: /1-133 Geminocystis herdmanii PCC 6308 (SEQ ID NO: 161) CLSYDTEVLTVEFGAIPMGKIVEERLNCQVYSVDKNGFIYTQNIAQWHDR GVQEVFEYELEDGRIIKATKDHKMMIENCEMVEIDRIFEEGLELFEVN (SEQ ID NO: 162) VKILKRRSISSQQVYDIGVEKDHNFLLANGLVASN >NnoPCC7104: /1-133 Nodosilinea nodulosa PCC 7104 (SEQ ID NO: 163) CLSASTELLTLEYGPLTIGEIVAKRIPCHVFSVDESGYVYTQPVAQWHQR GHQEVFEYQLDDGTTIRATIADHQFMTELGEMMAIDEIFQRGLELKQVE (SEQ ID NO: 164) VKIISRQSLGVQPVYDIGVARDHNFLLADGQVASN >RlaKORDI51-2: /1-137 Rubidibacter lacunae KORDI 51-2 (SEQ ID NO: 165) CLSYDTEVLTVEYGPLAIGTIVSERLACTVYTVDRSGFLYAQAISQWHER GRQDVFEYALDNGMTIRATKDHKLMTADGQMVAIDDIFTQGLTLKAIDTA AF (SEQ ID NO: 166) MKIVSRKSLGVQHVYDIGVARDHNFLLANGAIASN >CfrPCC9212/1-136 Chlorogloeopsis fritschii PCC 9212 (SEQ ID NO: 167) CLSYDTAILTVEYGFLPIGEIVEKGIECTVYTVDSNGYIYTQPIAQWHNR GEQELFEYSLEDGSIIRATKDHKFMTIDGQMLPIDEIFARKLELMQVKGL P (SEQ ID NO: 168) VKIIAKKSLGTQNVYDIGVERDHNFVIKNGLVASN >RinHH01: /1-137 Richelia intracellularis HH01 WGS project (SEQ ID NO: 169) CLSYDTQILTVEHGPMSIGEIVEKCLECHVYTVNKNGNICIQTITQWHFR GEQEIFEYELEDGSFIQATKDHKFMTTTGEMLPIHEIFTNGLEILQLSKS LL (SEQ ID NO: 170) VKILARKSLGTQKVYDIGVNDDHNFALSNSFIASN >SspPCC7117/1-137 (SEQ ID NO: 171) CLAGDTPVVTVEYGVLPIQTIVEQELLCQVYSVDAQGLIYTQPIEQWHNR GDRLLYEYELENGQMIRATPDHKFLTTTGELLPIDEIFTQNLDLAAWAVP DS (SEQ ID NO: 172) VKIIRRKFIGHAPTYDIGLSQDHNFLLGQGLIAAN >SspPCC8807/1-137 (SEQ ID NO: 173) CLAGDTPVVTVEYGVLPIQTIVEQELLCHVYSVDAQGLIYTQPIEQWHQR GDRFLYEYELENGQMIRATPDHKFLTTTGKLLPIDEIFTQNLDLAAWAVP DS (SEQ ID NO: 174) VKIIRRKFIGHAPTYDIGLSQDHNFLLGQGFIAAN >SspNKBG042902: /1-137 Synechococcus sp. NKBG 042902 (SEQ ID NO: 175) CLAGDTPVVTVEYGVLPIQTIVEQELLCHVYSVDAQGLIYTQPIEQWHQR GDRLLYEYELENGQMIRATPDHKFLTTTGELLPIDEIFTQNLDLAAWAVP DS (SEQ ID NO: 176) VKILRRKFIGRAPTYDIGLSQDHNFLLGQGLVAAN >SspNKBG15041: /1-129 Synechococcus sp. NKBG15041 (SEQ ID NO: 177) CLAGDTPVVTVEYGVLPIRTIVDQELLCHVYSLDPQGFIYAQPVEQWHRR GDRLLYEYELETGAVIRATPDHKFLTATGEMLPIDEIFVRNLDL (SEQ ID NO: 178) VKIIRRNLIGEAATYDIGLGKDHNFLLGQGLIASN >SspPCC73109/1-130 (SEQ ID NO: 179) CLAGGTPVVTVEYGVLPIQTIVEQELLCHVYSVDAQGLIYTQPIEQWHQR GDRLLYEYELENGQMIRATPDHKFLTTTGELLPIDEIFTQNLDLL (SEQ ID NO: 180) VKIIRRKFIGHAPTYDIGLSQDHNFLLGQGLIAAN >SspPCC7003/1-130 (SEQ ID NO: 181) CLAGDTPVVTVEYGVLPIQTIVEQELLCHVYSVDAQGLIYTQPIEQWHKR GDRLLYEYELENGQIIRATPDHKFLTTTGEMRPIDEIFAKNLSLL (SEQ ID NO: 182) VKIIRRKFVGHAPTYDIGLSQDHNFLLGQGLIAAN >CspPCC8802/ 1-134: Cyanothece sp. PCC 8802 (SEQ ID NO: 183) CLSYDTEILTVEYGAIPIGKVVEENIDCTVYTVDKNGFVYTQNIAQWHLR GQQEVFEYYLDDGSILRATKDHQFMTLEGEMLPIHEIFERGLELKKIKI (SEQ ID NO: 184) VKIVSYRSLGKQFVYDIGVAQDHNFLLANGSIASN >SelPCC7942: /1-137 Synechococcus elongatus PCC 7942 (SEQ ID NO: 185) CLAADTEVLTVEYGPIAIGKLVEENIRVQCYCCNPDGYIYSQPIGQWHQR GEQEVIEYELSDGRIIRATADHRFMTEEGEMLSLDEIFERSLELKQIPTP LL (SEQ ID NO: 186) VKIVRRRSLGVQPVYDLGVATVHNFVLANGLVASN >CfrPCC6912: 1-137 Chlorogloeposis fritschii PCC 6912 (SEQ ID NO: 187) CLSYDTAILTVEYGFLPIGEIVEKGIECTVYTVDSNGYIYTQPIAQWHNR GEQELFEYSLEDGSIIRATKDHKFMTIDGQMLPIDEIFARKLELMQVKGL PE (SEQ ID NO: 188) VKIIAKKSLGTQNVYDIGVERDHNFVIKNGLVASN >CspATC51472: /1-132 Cyanothece sp. ATCC 51472 (SEQ ID NO: 189) CLSYDTEILTVEYGPMPIGKIVEENINCTVYTVDPNGFVYTQAIAQWHYR GEQEIFEYYLEDGATIRATKDHKFMTMEGKMLPIDEIFENNLDLKQL (SEQ ID NO: 190) VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN >Lma: /1-132 Lyngbya majuscula (SEQ ID NO: 191) CLSYDTEIITVEYGPIAIGEIVEKGIPCTVYSVDSNGYVYTQPIAQWHNR GEQEVFEYTLDDGSVIRATKDHKFMTIDGQMLPIDEIFEGGLELKQL (SEQ ID NO: 192) VKIISRKSLGTQPVYDIGVKDDHNFILANGMVASN >CspESFC/1-137 (SEQ ID NO: 193) CLSYDTEVLTVEYGAVPIGKLVEEKLNCSVYTVDPNGYIYTQAIAQWHDR GIQEVFEYQLEDNTIIRATKDHKFMTEDHQMLPIDEIFERGLELKKCPQP QQ (SEQ ID NO: 194) VKIIRRRSLGFQPVYDIGLEQDHNFLLNQGAIASN >SspPCC7002: /1-129 Synechococcus sp. PCC 7002 (SEQ ID NO: 195) CLAGGTPVVTVEYGVLPIQTIVEQELLCHVYSVDAQGLIYAQLIEQWHQR GDRLLYEYELENGQMIRATPDHRFLTTTGELLPIDEIFTQNLDL (SEQ ID NO: 196) VKIIRRKFIGHAPTYDIGLSQDHNFLLGQGLIAAN >AmaMBIC11017: /1-132 Acaryochloris marina MBIC11017 (SEQ ID NO: 197) CLSYDTPVLTLEYGWLPIGQVVQEQIECQVFSINERGHLYTQPIAQWHHR GQQEVFEYTLADGSTIQATAEHQFMTTDGQMYPVQQIFEEGLSLKQL (SEQ ID NO: 198) VKIIQRRSLGLQSVYDIGLAQDHNFVMANGWVAAN >Mae905: /1-129 Microcystis aeruginosa DIANCHI905 (SEQ ID NO: 199) CLGGETLILTEEYGLLPIAKIVSEEVNCTVYSVDKNGFVYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTNDGEMLAIDTIFERGLDL (SEQ ID NO: 200) VKIISRQSLGRKPVYDIGVEKDHNFLLGNGLIASN >AciAWQC310F: /1-125 AWQC: Anabaena circinalis SWQC310F (SEQ ID NO: 201) CLSYDTEILTVEYGFLEIGEIVEKQIECKVYTVDSNGILYTQPIAQWHHR GQQEVYEYLLENGAIIRATKDHKFMTEAGEMLPIDDIFTQ (SEQ ID NO: 202) VKIISRTYVGQANVYDIGVENDHNFVIKNGFVAAN >AciAWQC131C: /1-125 Anabaena circinalis AWQC131C (SEQ ID NO: 203) CLSYDTEILTVEYGFLEIGEIVEKQIECRVYTVDSNGILYTQPIAQWHYR QQEVYEYLLENGAIIRATKDHNFMTEAGEMLPIDDIFTQ (SEQ ID NO: 204) IKIISRKYVGQANVYDIGVENDHNFVIKNGFVAAN >CspUCYN: /1-124 Cyanobacterium sp. UCYN-A2 (SEQ ID NO: 205) CLSYDTKVLTVEYGPLPIGKVVQENIRCRVYTTNDQGLIYTQPIAQWHNR GKQEIFEYHLDDKTIIRATKEHQFMTVDHVMMPIDEIFEQ (SEQ ID NO: 206) KIIRRKSLGMHEVFDIGLEKDHNFVLSNGLIASN >Pst: 1-129 Planktothrix st147: st147_cleanDRAFT_c6 (SEQ ID NO: 207) CLSYDTEVLTVEYGLIPISKIVEEKIECTVYTVNNQGYVYTQPIAQWHNR GEQEVFEYYLEDGSVIRATKDHKFMTVEGQMLPIDEIFEKELDL (SEQ ID NO: 208) VKIISRKSLGTQPVYDIGVQEDHNFVLNNGLVASN >PlaCYA98/1-129: Planktothrix NIVA-CYA 98 (SEQ ID NO: 209) CLSYDTEILTVEYGLMPIGKIVKEKIECTVYTVNNQGYVYTQPIAQWHHR GEQEVFEYCLEDGSVIRATKDHKFMTVQGQMLPIDEIFEKELDL (SEQ ID NO: 210) VKIISRKSLGTQPVYDIGVQEDHNFLLNNGLVASN >FdiUTEX481: 1-137 Fremyella diplosiphon UTEX 481 (SEQ ID NO: 211) CLSYDTEVLTVEYGLIPIGEIVEKRLECSVYSVDINGNVYTQPIAQWHHR GQQEVFEYALEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLLQVPHL PE (SEQ ID NO: 212) VKIVTRRAIGAANVYDIGVEQDHNFAIKNGLIAAN >Pst585: /1-129 Planktothrix sp. 585: Length = 1586997 (SEQ ID NO: 213) CLSYDTEILTVEYGLIPISKIVEEKIECTVYTVNNQGYVYTQPIAQWHNR GEQEVFEYYLEDGSVIRATKDHKFMTVDGQMLPIDEIFEKELDL (SEQ ID NO: 214) VKIISRKSLGTQPVYDIGVQEDHNFVLNNGLVASN >NpuPPC73102/1-137 (SEQ ID NO: 215) CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNL PN (SEQ ID NO: 216) IKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN >CthPCC7203: /1-137 Chrocococcidiopsis thermalis PCC 7203 (SEQ ID NO: 217) CLSYDTEILTVEYGAIPIGKIVEERIECTVYSVDNNGFIYTQPIAQWHNR GQQEVFEYCLEDGSIIRATKDHKFMTFEGKMLPIDEIFEQELDLKQVKSI QN (SEQ ID NO: 218) VKIISRKSLGIQPVYDIGVERDHKFVLKNGLVASN >NspCCY9414: /1-137 Nodularia spumigena CCY9414 genome (SEQ ID NO: 219) CLSYDTEILTVEYGYIPIGEIVEKAIECSVYSVDNNGNVYTQPIAQWHNR GEQEVFEYSLEDGSTIRATKDHKFMTTDGQMLPIDEIFAQELDLLQVHGL PK (SEQ ID NO: 220) VKITARKFVGRENVYDIGVERYHNFAIKNGLIASN >AcyPCC7122: /1-137 Anabaena cylindrica PCC 7122 (SEQ ID NO: 221) CLSYDTEVLTVEYGFIPIGEIVEKRIECSIFSVDKNGNVYTQPIAQWHNR GRQEIYEYCLDDGSKIRATKDHKFMTTAGEMLPIDEIFERDLDLLKVEGL PE (SEQ ID NO: 222) VKIISRQYLGQADVYDIGVEEDHNFAIKNGFIASN >CspPCC7507: /1-137 Calothrix sp. PCC 7507, complete genome (SEQ ID NO: 223) CLSYDTEVLTVEYGLLPIGEIVEKGIECRVFSVDNHGNVYTQPIAQWHNR GQQEVFEYGLDDGSVIRATKDHKFMTTDGKMLPIDEIFERGLDLLQVQGL PE (SEQ ID NO: 224) VKVITRKYIGKENVYDIGVELDHNFAIRNGLVASN >NspPCC7524: /1-137 Nostoc sp. PCC 7524 (SEQ ID NO: 225) CLSYDTEILTVEYGFLPIGEIVEKGIECTVFSVASNGIVYTQPIAQWHNR GQQEIFEYCLEDGSIIRATKDHKFMTQDGQMLPIDEIFACELDLLQVQGL PE (SEQ ID NO: 226) VKVVTRKYIGKENVYDIGVERDHNFVIRNGLVASN >Naz0708: /1-137 ‘Nostoc azollae’ 0708 (SEQ ID NO: 227) CLSYKTEVLTVEYGLIPIGEIVEKRIECSLFSVDENGNIYTQPIAQWHHR GVQEVYEYCLDDGTIIRATKDHKFMTTIGEMLPIDEIFERDLNLLQVNGL PT (SEQ ID NO: 228) VKIIRSQFLGPANVYDIGVAQDHNFAIKNGLIASN >NspPCC7120: /1-137 Nostoc sp. PCC 7120 DNA (SEQ ID NO: 229) CLSYDTEVLTVEYGFVPIGEIVEKGIECSVFSINNNGIVYTQPIAQWHHR GKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGL PE (SEQ ID NO: 230) IKIASRKFLGVENVYDIGVRRDHNFFIKNGLIASN >AvaATCC29413/1-137 Anabaena variabilis ATCC 29413 (SEQ ID NO: 231) CLSYDTEVLTVEYGFVPIGEIVDKGIECSVFSIDSNGIVYTQPIAQWHHR GKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGL PE (SEQ ID NO: 232) IKIASRKFLGVENVYDIGVGRDHNFFVKNGLIASN >PspPCC7327: /1-135 Pleurocapsa sp. PCC 7327. (SEQ ID NO: 233) CLSYDTKILTVEYGAMPIGKIVEEQIDCTVYTVNQNGFVYTQPIAQWHDR GKQEIFEYCLEDGSIIRATKDHKFMTTDGQMLPIDKIFEKGLDLKTINCD (SEQ ID NO: 234) VKILSRKSLGIQSVYDIGVEKDHNFLLANGLVASN >CspPCC7424: /1-135 Cyanothece sp. PCC 7424 (SEQ ID NO: 235) CLSYETQIMTVEYGLMPIGKIVEEQIDCTVYTVNKNGFVYTQPIAQWHYR GEQEVFEYCLEDGSTIRATKDHKFMTTDGQMLPIDEIFEQGLELKQIHLS (SEQ ID NO: 236) VKIISRQSLGIQPVYDIGVEKDHNFLISDGLIASN >CspPCC7822: /1-134 Cyanothece sp. PCC 7822 (SEQ ID NO: 237) CLSYDTEILTVEYGPMPIGKIVEEQIECTVYTVDKNGLVYTQPIAQWHHR GQQEVFEYCLEDGSIIRATKDHKFMTDDGQMLPIEEIFEKGLELKQIIL (SEQ ID NO: 238) VKIISRQLAGNQTVYDLGVEKDHNFLLANGLIASN >NspPCC7107: 1/137 Nostoc sp. PCC 7107 (SEQ ID NO: 239) CLSYDTQVLTVEYGLVPIGEIVEKQLECSVFTIDGHGYVYTQAIAQWHNR GQQEVFEYGLEDGSVIRATKDHKFMTTDGQMLPIDEIFERELDLLQVQGL RW (SEQ ID NO: 240) VKIITRKYIGQANVYDIGVAQDHNFVIENRLIASN >TboIicb1/1-136 Tolypothrix bouteillei Iicb1 (SEQ ID NO: 241) CLSYDTEILTVEYGFLPIGKIVEKGIECNVYSVDKNGNIYTQPIAQWHDR GEQEVFEYCLENGSVIRATKDHKFMTTSGEMLPIDEIFERGLDLIRVEDL P (SEQ ID NO: 242) VKILTRKSIGKQTVYDIGVERDHNFVIKNGSVASN >Aov: /1-136 Aphanizomenon ovalisporum DnaE precursor (dnaE) gene (SEQ ID NO: 243) CLSADTEILTVEYGFLPIGEIVGKAIECRVYSVDGNGNIYTQSIAQWHNR GEQEVFEYTLEDGSIIRATKDHKFMTTDGEMLPIDEXFARQLDLMQVQGL H (SEQ ID NO: 244) VKITARKFVGRENVYDIGVEHHHNFAIKNGLIASN >OnvPCC7112: /1-137 Oscillatoria nigro-viridis PCC 7112 (SEQ ID NO: 245) CLSYDTKILTVEYGPMAIGKIVEEKIECTVYSVDSNGYIYTQSIAQWHRR GQQEVFEYCLEDGSIIRATKDHKFMTVGGQMLPIDEIFEQGLDLKQINSS SD (SEQ ID NO: 246) VKIISRKSLGTQEVYDIGVEREHNFILENSLVASN >RspPCC7116: /1-135 Rivularia sp. PCC 7116, complete genome (SEQ ID NO: 247) CLSYDTEVLTEEFGLIPIGKIVEEKIDCTVYSVDVNGNVYSQPIAQWHNR GMQEVFEYELEDGSTIRATKDHKFMTVDGEMLAIDEIFEKGLELKRVGIY (SEQ ID NO: 248) VKIISRKVLKTENVYDIGLEGDHNFIIKDGLIASN >MspPCC7113: /1-137 Microcoleus sp. PCC 7113, (SEQ ID NO: 249) CLSYDSEILTVEYGLMPIGKIVEEGIECTVYSVDSHGYLYTQPIAQWHHR GQQEVFEYDLEDGSVIRATKDHKFMTSEGQMLAIDEIFERGLELKQVKRS QP (SEQ ID NO: 250) VKIVRRKSLGIQTVYDIGVERDHNFLLANGLVASN >ScyPCC7437: /1-137 Stanieria cyanosphaera PCC 7437 (SEQ ID NO: 251) VLSYDTEILTVEYGAMPIGKIVKEQIECNVYTVNQNGFIYPQAIAQWHER GKQEIFEYTLDNGLVIRATKDHKFMTIDGQMLPIDEIFERGLELQRINDY SN (SEQ ID NO: 252) VKIVSRKSLGKQPVYDIGVTKDHNFLLSNGVVASN >CspPCC6303: /1-137 Calothrix sp. PCC 6303 (SEQ ID NO: 253) CLSYDTEILTWEYGFLKIGEIVEKQILCSVFSVDEQGNVYTQPIAQWHNR GLQELFAYQLEDGGVIRATKDHKFMTTDGQMLAIDEIFERQLDLFQVKGL PE (SEQ ID NO: 254) VKIISRKVLKTENVYDIGLEGDHNFIIDGLIASN >Cst: /1-134 PCC7202: Cyanobacterium stanieris PCC 7202 (SEQ ID NO: 255) CLSYDTEVLTVEYGVLPIGKIVEEQIQCTVYSVDQYGFVTYQAIAQWHDR GEQEVFEYELENGATIKATKDHKMMTSDGQMLPIDQIFEQGLDLFMVSF (SEQ ID NO: 256) VKIVKRRSHGIQKVYDIGVAKDHNFLLHNGLVASN >CspATCC51142: /1-134 Cyanothece sp. ATCC 51142 (SEQ ID NO: 257) CLSYDTEILTVEYGPMPIGKIVEENINCTVYTVDPNGFVYTQAIAQWHYR GEQEIFEYYLEDGATIRATKDHKFMTMEGKMLPIDEIFENNLDLKQLTL (SEQ ID NO: 258) VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN >CspPCC8801: /1-134 Cyanothece sp. PCC 8801 (SEQ ID NO: 259) CLSYDTEILTVEYGAIPIGKVVEENIDCTVYTVDKNGFVYTQNIAQWHLR GQQEVFEYYLDDGSILRATKDHQFMTLEGEMLPIHEIFERGLELKKIKI (SEQ ID NO: 260) VKIVSYRSLGKQFVYDIGVAQDHNFLLANGSIASN >Asp: /1-136 Anabaena sp. 90 chromosome (SEQ ID NO: 261) CLSYDTEILTVEYGFLEIGEIVEKQIECKVYTIDSNGMLYTQSIAQWHNR GQQEVYEYLLENGAIIRATKDHKFMTEAGQMLPIDEIFAQGLDLLQVGVA E (SEQ ID NO: 262) VKIVSRTYVGQANVYDIGVESDHNFVIKNGFIASN >Aha: /1-137 Aphanothece halpphytica (SEQ ID NO: 263) CLSYDTEIWTVEYGAMPIGKIVEEKIECSVYTVDENGFVYTQPIAQWHPR GQQEIIEYTLEDGRKIRATKDHKMMTESGEMLPIEEIFQRELDLKVETFH EM (SEQ ID NO: 264) VKIIKRQSLGRQNVYDVCVETDHNFVLANGCVASN >HspPCC7418: /1-137 Halothece sp. PCC 7418 (SEQ ID NO: 265) CLSYDTEIWTVEYGAMPIGKIVEEKIECSVYTVDENGFVYTQPIAQWHPR GQQEIIEYTLEDGRKIRATKDHKMMTESGEMLPIEEIFQRELDLKVETFH EM (SEQ ID NO: 266) VKIIKRQSLGRQNVYDIGVETDHNFVLANGCVASN >CapPCC10605: /1-137 Cyanobacterium aponium PCC 10605 (SEQ ID NO: 267) CLSYDTEILTVEYGAISIGKIVEEKINCQVYSVDKNGFIYTQNIAQWHDR GSQELFEYELEDGRIIKATKDHKMMTKDGQMLAINDIFEQELELYSVDDM GV (SEQ ID NO: 268) VKIVKRRSLGVQPVYDIGVEKDHNFILANGLVASN >Cat: /1-133 Candidatus Atelocyanobacterium thalassia isolate (SEQ ID NO: 269) CLSYDTKVLTVEYGPLPIGKVVQENIRCRVYTTNDQGLIYTQPIAQWHNR GKQEIFEYHLDDKTIIRATKEHQFMTVDHVMMPIDEIFEQGLELKKIK (SEQ ID NO: 270) LKIIRRKSLGMHEVFDIGLEKDHNFVLSNGLIASN <Oli: /1-137 Oscillatoria limnetica ‘Solar Lake’ DnaE precursor (SEQ ID NO: 271) CLSYNTEVLTVEYGPLPIGKIVDEQIHCRVYSVDENGFVYTQAIAQWHDR GYQEIFAYELADGSVIRATKDHQFMTEDGQMFPIDEIWEKGLDLKKLPTV QD (SEQ ID NO: 272) VKIVRRQSLGVQNVYDIGVEKDHNFLLASGEIASN >Cen: /1-137 Cyanobacterium endosymbiont of Epithemia turgida (SEQ ID NO: 273) CLSYDTEVLTVEYGAIPIGRMVEESLDCTVYTVDKNGFVYTQSIQQWHSR GQQEIFEYCFEDGSIIRATKDHKFMTAEGKMSSIHDIFEQGLELKKIIPW SG (SEQ ID NO: 274) AKIISCKSLGKQSVYDIGVVQDHNFLLANGVVASN >SspPCC7502: /1-133 Synechococcus sp. PCC 7502 CLGYDTPVLTVEYGFMPIGKIVEEKIQCHVYSVDQNGLVFTQAIAQWHNR GQQEVWEYNLDNGDIVRATKDHKFMTIDGQMLPINQIFEQGLELKVIA (SEQ ID NO: 276) VKIVSCKPLRVQTVYDIGVEKDHNFILDNGLVASN >CspUCYN: /1-124 Cyanobacterium sp. UCYN-A2 (SEQ ID NO: 277) CLSYDTKVLTVEYGPLPIGKVVQENIRCRVYTTNDQGLIYTQPIAQWHNR GKQEIFEYHLDDKTIIRATKEHQFMTVDHVMMPIDEIFEQ (SEQ ID NO: 278) KIIRRKSLGMHEVFDIGLEKDHNFVLSNGLIASN >Pst: /1-129 Planktothrix st147: st147_cleanDRAFT_c6 (SEQ ID NO: 279) CLSYDTEVLTVEYGLIPISKIVEEKIECTVYTVNNQGYVYTQPIAQWHNR GEQEVFEYYLEDGSVIRATKDHKFMTVEGQMLPIDEIFEKELDL (SEQ ID NO: 280) VKIISRKSLGTQPVYDIGVQEDHNFVLNNGLVASN >PlaCYA98/1-129: Planktothrix NIVA-CYA 98 (SEQ ID NO: 281) CLSYDTEILTVEYGLMPIGKIVKEKIECTVYTVNNQGYVYTQPIAQWHHR GEQEVFEYCLEDGSVIRATKDHKVMTVQGQMLPIDEIFEKELDL (SEQ ID NO: 282) VKIISRKSLGTQPVYDIGVQEDHNFLLNNGLVASN >Pst585: /1-129 Planktothris sp. 585: Length = 1586997 (SEQ ID NO: 283) CLSYDTEILTVEYGLIPISKIVEEKIECTVYTVNNQGYVYTQPIAQWHNR GEQEVFEYYLEDGSVIRATKDHKFMTVDGQMLPIDEIFEKELDL (SEQ ID NO: 284) VKIISRKSLGTQPVYDIGVQEDHNFVLNNGLVASN >CspPCC8802/ 1-134: Cyanothece sp. PCC 8802 (SEQ ID NO: 285) CLSYDTEILTVEYGAIPIGKVVEENIDCTVYTVDKNGFVYTQNIAQWHLR GQQEVFEYYLDDGSILRATKDHQFMTLEGEMLPIHEIFERGLELKKIKI (SEQ ID NO: 286) VKIVSYRSLGKQFVYDIGVAQDHNFLLANGSIASN >CfrPCC6912: /1-137 Chlorogloeposis fritschii PCC 6912 (SEQ ID NO: 287) CLSYDTAILTVEYGFLPIGEIVEKGIECTVYTVDSNGYIYTQPIAQWHNR GEQELFEYSLEDGSIIRATKDHKFMTIDGQMLPIDEIFARKLELMQVKGL PE (SEQ ID NO: 288) VKIIAKKSLGTQNVYDIGVERDHNFVIKNGLVASN >CspATC51472: /1-132 Cyanothece sp. ATCC 51472 (SEQ ID NO: 289) CLSYDTEILTVEYGPMPIGKIVEENINCTVYTVDPNGFVYTQAIAQWHYR GEQEIFEYYLEDGATIRATKDHKFMTMEGKMLPIDEIFENNLDLKQL (SEQ ID NO: 290) VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN >Lma: /1-132 Lyngmya majuscula (SEQ ID NO: 291) CLSYDTEIITVEYGPIAIGEIVEKGIPCTVYSVDSNGYVYTQPIAQWHNR GEQEVFEYTLDDGSVIRATKDHKVMTIDGQMLPIDEIFEGGLELKQL (SEQ ID NO: 292) VKIISRKSLGTQPVYDIGVKDDHNFILANGMVASN >CspESFC/1-137 (SEQ ID NO: 293) CLSYDTEVLTVEYGAVPIGKLVEEKLNCSVYTVDPNGYIYTQAIAQWHDR GIQEVFEYQLEDNTIIRATKDHKFMTEDHQMLPIDEIFERGLELKKCPQP QQ (SEQ ID NO: 294) VKIIRRRSLGFQPVYDIGLEQDHNFLLNQGAIASN >Mae905: /1-129 Microcystis aeruginosa DIANCHI905 (SEQ ID NO: 295) CLGGETLILTEEYGLLPIAKIVSEEVNCTVYSVDKNGFVYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTNDGEMLAIDTIFERGLDL (SEQ ID NO: 296) VKIISRQSLGRKPVYDIGVEKDHNFLLGNGLIASN >RlaKORDI51-2: /1-137 Rubidibacter lacunae KORDI 51-2 (SEQ ID NO: 297) CLSYDTEVLTVEYGPLAIGTIVSERLACTVYTVDRSGFLYAQAISQWHER GRQDVFEYALDNGMTIRATKDHKLMTADGQMVAIDDIFTQGLTLKAIDTA AF (SEQ ID NO: 298) MKIVSRKSLGVQHVYDIGVARDHNFLLANGAIASN >CfrPCC9212/1-136 Chlorogloeopsis fritschii PCC 9212 (SEQ ID NO: 299) CLSYDTAILTVEYGFLPIGEIVEKGIECTVYTVDSNGYIYTQPIAQWHNR GEQELFEYSLEDGSIIRATKDHKFMTIDGQMLPIDEIFARKLELMQVKGL P (SEQ ID NO: 300) VKIIAKKSLGTQNVYDIGVERDHNFVIKNGLVASN >RinHH01: /1-137 Richelia intracellularis HH01 WGS project (SEQ ID NO: 301) CLSYDTQILTVEHGPMSIGEIVEKCLECHVYTVNKNGNICIQTITQWHFR GEQEIFEYELEDGSFIQATKDHKFMTTTGEMLPIHEIFTNGLEILQLSKS LL (SEQ ID NO: 302) VKILARKSLGTQKVYDIGVNDDHNFALSNSFIASN >GhePCC6308: /1-133 Geminocystis herdmanii PCC 6308 (SEQ ID NO: 303) CLSYDTEVLTVEFGAIPMGKIVEERLNCQVYSVDKNGFIYTQNIAQWHDR GVQEVFEYELEDGRIIKATKDHKMMIENCEMVEIDRIFEEGLELFEVN (SEQ ID NO: 304) VKILKRRSISSQQVYDIGVEKDHNFLLANGLVASN >SsuPCC9445: /1-131 Spirulina subsalsa PCC 9445 (SEQ ID NO: 305) CLSYDTKIITVEYGAIAIGTIVEQGLHCHVYSVDPNGFIYTQPIAQWHQR GEQEVFAYTLENGSIIQATKDHKFMTQQGKMLPIDTIFEQGLDLLQV (SEQ ID NO: 306) KIIKRTSLGVRPVYDIGVIQDHNFLLENGLVASN >MaePCC9807: /1-135 Microcystis aeruginosa 9807 (SEQ ID NO: 307) CLGGETLILTEEYGLLPIAKIVSEEINCTVYSVDKNGFIYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTSDGEMLAIDTIFERGLDLKSSDFS (SEQ ID NO: 308) VKIISRQFLGRKPVYDIGVEKDHNFLLGNGLIASN >MspGI1: /1-130 Myxosarcina sp. GI1 contig_13 (SEQ ID NO: 309) CLSYDTEVLTLKYGALPIGEIVEKRINCHVYTRAESGFFYIQSIEQWHDR GEQEVFEYTLENGATIKATKDHKFMTSGGQMLPIDEIFERGLDLL (SEQ ID NO: 310) VKIVSRKSLGKQPVYDLGVAKDHNFLLANGTVASN >ShoPCC7110: /1-136 Scytonema hofmanni PCC 7110 contig00136 (SEQ ID NO: 311) CLSYDTEVLTAEYGFLPIGKIVEKAIECTVYSVDNDGNIYTQPIAQWHDR GQQEVFEYSLDDGSVIRATKDHKFMTTGGQMLPIDEIFERGLDLMRIDSL P (SEQ ID NO: 312) VKILTRKSIGKQTVYDIGVERDHNFVIKNGLVASN >WinUHHT291/1-136 Westiella intrica UH HT-29-1 (SEQ ID NO: 313) CLSYDTEILTVEYGFLPIGEIVEKRIECTVYTVDTNGYVYTQAIAQWHNR GEQEVFEYALEDGSIIRATKDHKFMTSEGQMLPIDEIFVKGLDLLQVQGL P (SEQ ID NO: 314) VKIITRKFLGIQNVYDIGVEQNHNFVIKNGLVASN >FspPCC9605: /1-136 Fischerella sp. PCC 9605 FIS9605DRAFT (SEQ ID NO: 315) CLSYDTEILTVEYGFLPIGEIVEKGIECTVYTVDNNGNVYTQTIAQWHNR GQQEVFEYCLEDGSVIRATKDHKFMTTDGQMLPIDEIFARGLDLLQVKNL P (SEQ ID NO: 316) VKIVTRRPLGTQNVYDIGVESDHNFVIKNGLVASN >MrePCC10914: /1-137 Mastigocladopsis repens PCC 10914 (SEQ ID NO: 317) CLSYDTEVLTVEYGFLPIGEIVEKSIECSVYTVDSNGNVYTQPIAQWHNR GQQEVFEYCLEDGSIIRATKDHKFMTIHGQMLPIDEIFERGLELMKIQGL PE (SEQ ID NO: 318) AKIITRKSLGTQNVYDIGVERDHNFVTRDGFIASN >ShoUTEX2349: /1-137 [Scytonema hofmanni] UTEX 2349 (SEQ ID NO: 319) CLSYNSEVLTVEYGFLPIGKIVEKGIECSVYSVDSYGKIYTQVIAQWHNR GQQEVFEYCLEDGTIIQATKDHKFMTVDGQMLPIDEIFERGLDLMQVQGL PD (SEQ ID NO: 320) VKIITRKSLGTQNVYDIGVSSDHNFVMKNGLIASN >AspPCC7108: /1-137 Anabaena sp. PCC 7108 Ana7108scaffold_2_Cont3 (SEQ ID NO: 321) CLSSDTEVLTVEYGLIPIGEIIEKRIDCSVFSVDKNGNIYTQPIAQWHDR GIQELYEYCLDDGSTIRATKDHKFMTTAGEMLPIDEIFERGLDLLKVHNL PQ (SEQ ID NO: 322) VKIITRNYVGKENVYDIGVERDHNFAIKNGLIASN >FspPCC9339: /1-137 Fischerella sp. PCC 9339 PCC9339DRAFT (SEQ ID NO: 323) CLSYDTEVLTVEYGFLPIGEIVEKRIECTVYTVDHNGYVYTQPIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTSEGQMLPIDEIFARELDLLQVTGL VN (SEQ ID NO: 324) VKIVTRRLLGIQNVYDIGVEQNHNFVIKNGLVASN >Csp336: /1-137 Calothrix sp. 336/3 (SEQ ID NO: 325) CLSYDTEIFTVEYGFLPIGEIVEKRLECTVLTVDNHGNIYSQPIAQWHHR GQQQQIEYEGLEDGSVIRATKDHKFMTTDGQMLPIDEIFERGLDLLQVTN LDN (SEQ ID NO: 326) VKVITRKLADTENVYDIGVENHHNFLIKNGLVASN >FthPCC7521: /1-136 Fischerella thermalis PCC 7521 (SEQ ID NO: 327) CLSYETEILTVEYGFLPIGEIVEKRIECSVYTVDNNGYVCTQPIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTIDRQMLPIDEIFARGLDLLQVTGL P (SEQ ID NO: 328) VKIITRKSLGTQNVYDIGVEQNHNFVIKNGLVASN >CyaPCC7702/1-137 cyanobacterium PCC 7702 Chl7702 (SEQ ID NO: 329) CLSYDTEILTVEYGFLSIGEIVEKEIECTVYTVDSNGYIYTQPIAQWHEQ GEQEIFEYSLEDGSTIRATKDHKFMTIEGEMLPIDQIFARQLDLMQITGL PQ (SEQ ID NO: 330) VKISTKKSLGKQKVYDIGVVRDHNFIIKNGFVASN >FspPCC9431: /1-136 Fischerella sp. PCC 9431 (SEQ ID NO: 331) CLSYDTEVLTVEYGFLPIGEIVEKRIECTVYTVDTNGYVYTQAIAQWHNR DEQEVFEYALEDGSIIRATKDHKFMTSEGQMLPIDEIFAKGLDLLQVQGL P (SEQ ID NO: 332) VKIVTRKFLGIQNVYDIGVEQNHNFVIKNGLVASN >FmuPCC7414: /1-137 Fischerella muscicola PCC 7414 (SEQ ID NO: 333) CLSYETEILTVEYGFLPIGEIVEKRIECSVYTVDNNGYVCTQTIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTIDRQMLPIDEIFARGLDLLQVKGL PE (SEQ ID NO: 334) VKIITRQSLGTQNVYDIGVEQNHNFVIKNGLVASN >FmuPCC73103: /1-137 Fischerella muscicola SAG 1427-1 = PCC 73103 (SEQ ID NO: 335) CLSYDTEVLTVEYGFLPIGEIVEKTIECNVFTVDSNGYVYTQPIAQWHNR GYQEVFEYGLEDGSVIRATKDHKFMTSEGKMLPIDEIFARELDLLQVTGL IN (SEQ ID NO: 336) VKIVTRKFLGIQNVYDIGVEQNHNFVIKNGLVASN >Lae: /1-137 Lyngbya aestuarii BL J laest3.contig.3 (SEQ ID NO: 337) CLSYDTEILTVEYGAIPIGKVVDEKIECTVYSVDKNGLIYTQPIAQWHNR GKQEVFEYSLEDGSTIRATKDHKFMTMDNQMLPIDEILEKGLELKQVNAD SV (SEQ ID NO: 338) VKIVSRKSLDSQTVYDIGVETDHNFLLANGSVASN >Lsp: /1-137 Leptoyngbya sp. JSC-1 (SEQ ID NO: 339) CLSYDTEILTVEYGALPIGKIVENQMICSVYSIDNNGYIYIQPIAQWHNR GQQEVFEYILEDGSIIRSTKDHKFMTKGGEMLPIDEIFERGLELAQVTRL EQ (SEQ ID NO: 340) VKIISRRSVGVQSVYDIGVKQDHNFFLRNGLIASN >CwaWH8501: /1-137 Crocosphaera watsonii WH8501 (SEQ ID NO: 341) CLSYDTEILTVEYGAMYIGKIVEENINCTVYTVDKNGFVYTQTIAQWHNR GEQEIFEYDLEDGSKIKATKDHKFMTIDGEMLPIDEIFEKNLDLKQVVSH PD (SEQ ID NO: 342) VKIIGCRSLGTQKVYDIGVEKDHNFLLANGSIASN >CchPCC7420: /1-135 Coleofasciculus chthonoplastes PCC 7420 (SEQ ID NO: 343) CLSYDTQILTVEYGAVAIGEIVEKQIECTVYSVDENGYVYTQPIAQWHNR GEQEVFEYLLEDGATIRATKDHKFMTDEDQMLPIDQIFEQGLELKQVEVL (SEQ ID NO: 344) VKIIGRKPLGTQPVYDIGVERDHNFLLFNGSVASN >CspPCC6712/1-133 (SEQ ID NO: 345) CLSYDTEVLTVEYGAIPIGKIVEEKIACNVYSVDKNGFVYTQPIAQYHDR GIQEVFEYRLENGSVIRATKDHKMMTADGQMLPIDEIFKQNLDLKQLN (SEQ ID NO: 346) VKIISRQSLGKQSVFDIGVAKDHNFLLANGLVASN >Rbr: /1-137 Raphidiopsis brookii D9 D9_5, (SEQ ID NO: 347) CLSYETEVLTLEYGFLPIGEIVDKQMVCTVFSVNDSGNVYTQPIGQWHDR GVQELYEYCLDDGSTIRATKDHKFMTTQGEMVPIDEIFHQGWELVQVSGT MN (SEQ ID NO: 348) VKIVSRRYLGKADVYDIGVAKDHNFIIKNGLVASN >CspCCy0110: /1-134 Cyanothece sp. CCY0110 1101676644064 (SEQ ID NO: 349) CLSYDTEILTVEYGPMPIGKIVEENINCSVYTVNKNGFVYTQSIAQWHHR GEQEVFEYYLEDGETIRATKDHKFMTTEGKMLPIDEIFENNLDLKKLTV (SEQ ID NO: 350) VKIIERRSLGKQNVYDIGVEKDHNFLLSNNLIASN >XspPCC7305: /1-135 Xenococcus sp. PCC 7305 (SEQ ID NO: 351) CLSADTEVLTVEYGAISIGKIVEERIECTVYSVDANGFVYTQEIAQWHNR GEQEVFEYMLDDGSVIRATKDHKLMTIDGQMVAIDEIFSQGLELKQVLGL (SEQ ID NO: 352) VKIVSRKSLGTQTVYDLGVARDHNFLLANGTVASN <PspPCC7319: /1-135 Pleurocapsa sp. PCC 7319 (SEQ ID NO: 353) CLSYDTEIYTVEYGALPIGKIVESRIKCTVLTVDKNGLVYSQPIVQWHDR GIQEVFEYTLDNGATIRATKDHKFMTVEGQMLPIDEIFELGLELKEIQQF (SEQ ID NO: 354) VKIISRQSLGKQSVYDIGVAKDHNFLLANGMVASN >CraCS505: /1-137 Cylindrospermopsis raciborskii CS-505 (SEQ ID NO: 355) CLSYETEVLTLEYGFVPIGEIVNKQMVCTVFSLNDSGNVYTQPIGQWHDR GVQDLYEYCLDDGSTIRATKDHKFMTTQGEMVPIDEIFHQGWELVQVSGI SK (SEQ ID NO: 356) VKIVSRRYLGKADVYDIVAKDHNFIIKNGLVASN >MaePCC7806: /1-135 Microcystis aeruginosa PCC 7806 (SEQ ID NO: 357) CLGGETLILTEEYGLLPIAKIVSEEVNCTVYSVDKNGFVYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTNDGEMLAIDTIFERGLDLKSSDFS (SEQ ID NO: 358) VKIISRQSLGRKPVYDIGVEKDHNFLLGNGLIASN >MaeNIES843: /1-135 Microcystis aerugonosa NIES-843 DNA (SEQ ID NO: 359) CLGGETLILTEEYGLLPIAKIVSEEINCTVYTVDQNGFVYSQPISQWHER GLQEVFEYTLENGQTIQATKDHKFMTSDGEMLAIDTIFERGLDLKSSDFS (SEQ ID NO: 360) VKIIGRQSLGRKPVYDIGVEKDHNFLLGNGLIASN
(67)
(68) The Cfa intein has high sequence similarity to Npu (82%), and the non-identical residues are spread throughout the 3D structure of the protein.
(69) Cfa intein fragments fused to model exteins were generate and their PTS activity was measured using the aforementioned in vitro assay (
(70) Applications of PTS typically require fission of a target protein and fusion of the resulting fragments to the appropriate split intein segments..sup.1 As a consequence, the solubility of these fusion proteins can sometimes be poor. Because protein denaturants such as guanidine hydrochloride (GuHCl) and urea are frequently used to keep these less soluble fragments in solution, the ability of Cfa to splice in the presence of these chaotropic agents was tested. Cfa intein was found to splice in the presence of up to 4M GuHCl (with little decrease in activity seen up to 3M), while no activity was observed for Npu in ≥3M GuHCl (
(71)
(72) The unprecedented and unexpected tolerance of Cfa to high concentrations of GuHCl and urea suggests the intein might retain activity directly following chaotropic extraction of insoluble proteins from bacterial inclusion bodies, thereby expediting PTS-based studies. Accordingly, the model fusion protein, His.sub.6-Sumo-Cfa.sup.N, was overexpressed in E. coli cells and extracted the protein from inclusion bodies with 6M urea. The protein was purified from this extract by nickel affinity chromatography and then directly, and efficiently, modified by PTS under denaturing conditions, i.e. without the need for any intervening refolding steps. In general, it is expected that the robust activity of Cfa in the presence of chaotropic agents will prove useful when working with protein fragments that demonstrate poor solubility under native conditions.
(73) Fusing a protein of interest to a split intein can result in a marked reduction in cellular expression levels compared to the protein alone..sup.6 This situation is more frequently encountered for fusions to N-inteins than to C-inteins, which is likely due to the larger size of the former and their partially folded state..sup.18 It was therefore investigated whether the improved thermal and chaotropic stability of Cfa would translate to increased expression levels of Cfa.sup.N fusions. Indeed, model studies in E. coli revealed a significant (30-fold) increase in soluble protein expression for a Cfa.sup.N fusion compared to the corresponding Npu.sup.N fusion (
(74)
(75) Finally, to further explore the utility of the Cfa intein in the context of antibody conjugation, whether the PTS system could be used to attach multiple copies of a synthetic cargo to the heavy chain of the mAb was investigated. Accordingly, semisynthesis was used to prepare a construct in which the C-terminal half of Cfa (Cfa.sup.C) was fused to a C-extein containing a dendrimeric scaffold allowing multimeric attachment of cargo, in this case fluorescein (
(76)
(77) The discovery of fast split inteins has revolutionized the applications of protein trans splicing. The remarkable robustness of the Cfa intein described in this study should extend the utility of many of these technologies by allowing PTS to be performed in a broader range of reaction conditions. Moreover, the ability of Cfa to increase the expression yields of N-intein fusions should encourage further use of split inteins for protein semisynthesis. The activity-guided approach we use to engineer this intein may be applied to other intein families or act as a general strategy for the refinement of multiple sequence alignments used for consensus engineering.
(78) Materials and Methods
(79) Materials
(80) Oligonucleotides and synthetic genes were purchased from Integrated DNA Technologies (Coralville, Iowa). The QuickChange XL II site directed mutagenesis kit and Pfu Ultra II Hotsart fusion polymerase were purchased from Agilent (La Jolla, Calif.). All restriction enzymes and 2× Gibson Assembly Master Mix were purchased from New England Biolabs (Ipswich, Mass.). “In-house”high-competency cells used for cloning and protein expression were generated from One Shot Bl21 (DE3) chemically competent E. coli and sub-cloning efficiency DH5α competent cells purchased from Invitrogen (Carlsbad, Calif.). Dulbecco's Modified Eagle Medium (DMEM), Lipofectamine 2000, and low IgG fetal bovine serum were purchased from Invitrogen as well. DNA purification kits were purchased from Qiagen (Valencia, Calif.). All plasmids were sequenced by GENEWIZ (South Plainfield, N.J.). N,N-diisopropylethylamine (DIPEA), Luria Bertani (LB) media, and all buffering salts were purchased from Fisher Scientific (Pittsburgh, Pa.). Dimethylformamide (DMF), dichloromethane (DCM), Coomassie brilliant blue, triisopropylsilane (TIS), β-Mercaptoethanol (BME), DL-dithiothreitol (DTT), sodium 2-mercaptoethanesulfonate (MESNa), tetrakis(triphenylphosphine)palladium(0) (Pd(PPh.sub.3).sub.4), and 5(6)-carboxyfluorescein were purchased from Sigma-Aldrich (Milwaukee, Wis.) and used without further purification. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and isopropyl-β-D-thiogalaetopyranoside (IPTG) were purchased from Gold Biotechnology (St. Louis, Mo.). The protease inhibitor used was the Roche Complete Protease Inhibitor (Roche, Branchburg, N.J.). Nickel-nitrilotriacetic acid (Ni-NTA) resin was purchased from Thermo scientific (Rockford, Ill.). Fmoc amino acids were purchased from Novabiochem (Darmstadt, Germany) or Bachem (Torrance, Calif.). (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Genscript (Piscataway, N.J.). Rink Amide-ChemMatrix resin was purchased from Biotage (Charlotte, N.C.). Trifluoroacetic acid (TFA) was purchased from Halocarbon (North Augusta, S.C.). Immun-blot PVDF membrane (0.2 μm) and Criterion XT Bis-Tris gels (12% polyacrylamide) were purchased from Bio-Rad (Hercules, Calif.). MES-SDS running buffer was purchased from Boston Bioproducts (Ashland, Mass.). Anti-Mouse IgG secondary antibody (Licor mouse 800) and Mouse uActin primary antibody were purchased from Li-COR biotechnology (Lincoln, Nebr.).
(81) Equipment
(82) Analytical RP-HPLC was performed on Hewlett-Packard 1100 and 1200 series instruments equipped with a C.sub.18 Vydac column (5 μm, 4.6×150 mm) at a flow rate of 1 mL/min. Preparative RP-HPLC was performed on a Waters prep LC system comprised of a Waters 2545 Binary Gradient Module and a Waters 2489 UV detector. Purifications were carried out on a C.sub.18 Vydac 218TP1022 column (10 μM; 22×250 mm) at a flow rate of 18 mL/min. All runs used 0.1% TFA (trifluoroacetic acid) in water (solvent A) and 90% acetonitrile in water with 0.1% TFA (solvent B). Unless otherwise stated, peptides and proteins were analyzed using the following gradient: 0% B for 2 minutes (isocratic) followed by 0-73% B over 30 minutes. Electrospray ionization mass spectrometric analysis (ESI-MS) was performed on a Bruker Daltonics MicroTOF-Q II mass spectrometer. Size-exclusion chromatography was carried out on an AKTA FPLC system (GE Healthcare) using a Superdex S75 16/60 (CV=125 mL) column. Coomassie-stained gels and western blots were imaged using a LI-COR Odyssey Infrared Imager. Fluorescent gels were imaged using a GE ImageQuant LAS 4000 Imager. The splicing-dependent E. coli growth assay was performed on a VersaMax tunable microplate reader from Molecular Devices. Cell lysis was carried out using a S-450D Branson Digital Sonifier.
(83) Cloning of DNA Plasmids
(84) All N-intein constructs for E. coli expression were cloned into previously used pET and pTXB1 vectors..sup.1 Plasmids encoding for WT pet30-His.sub.6-SUMO-AEY-Ssp.sup.N, pet30-His.sub.6-SUMO-AEY-Npu.sup.N, pTXB1-Ssp.sup.C-MxeGyrA-His.sub.6, and pTXB1-Npu.sup.C-MxeGyrA-His.sub.6 plasmids were cloned as previously described.sup.1 and encode for the following protein sequences. Protein products after either SUMO cleavage (N-inteins) or thiolysis (C-inteins) are shown in bold for all plasmids.
(85) TABLE-US-00026 Plasmid 1: WT Ssp.sup.N: pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (SEQ ID NO: 361) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHRFLTTDYQLL AIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 2: WT Npu.sup.N: pet30-His.sub.6-SUMO-AEY-Npu.sup.N (SEQ ID NO: 362) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYALSYETEILTVEYGLLPIGKIVEKRIECTVY SVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQML PIDEIFERELDLMRVDNLPN Plasmid 3: WT Ssp.sup.C: pTXB1-Ssp.sup.C-MxeGyrA-His.sub.6 (SEQ ID NO: 363) MVKVIGRRSLGVQRIFDIGLPQDHNFLLANGAIAANCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH Plasmid 4: WT Npu.sup.C: pTXB1-Npu.sup.C-MxeGyrA-His.sub.6 (SEQ ID NO: 364) MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH
(86) All Ssp.sup.N batch mutants were cloned using the QuikChange site directed mutagenesis kit using plasmid 1 as a template and encode for the protein sequences shown below. The N-intein sequence is shown in bold with the residues corresponding to the batch mutation underlined.
(87) TABLE-US-00027 Plasmid 5: Batch 1: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (R73K, L75M, Y79G, L81M) (SEQ ID NO: 365) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHKFMTTDGQML AIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 6: Ssp.sup.N R73K: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (R73K) (SEQ ID NO: 366) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHKFLTTDYQLL AIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 7: Ssp.sup.N R73K Y79G: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (R73K, Y79G) (SEQ ID NO: 367) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHKFLTTDGQLL AIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 8: Ssp.sup.N R73K Y79G L81M: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (R73K, Y79G, L81M) (SEQ ID NO: 368) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHKFLTTDGQML AIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 9: Batch 2: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (L56F, S70K, A83P, E85D) (SEQ ID NO: 369) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVFEYELEDGSVIRATKDHRFLTTDYQLL PIDEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 10: Ssp.sup.N A83P: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (A83P) (SEQ ID NO: 370) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHRFLTTDYQLL PIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 11: Ssp.sup.N S70K A83P: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (S70K, A83P) (SEQ ID NO: 371) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATKDHRFLTTDYQLL PIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 12: Ssp.sup.N L56, S70K, A83P: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (L56F, S70K, A83P) (SEQ ID NO: 372) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDPEGRVYTQAIAQWHDRGEQEVFEYELEDGSVIRATKDHRFLTTDYQLL PIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 13: Batch 3: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (523E, E24K, E25R, N27E) (SEQ ID NO: 373) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVEKRIECSVY SVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHRFLTTDYQLL AIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK Plasmid 14: Batch 4: Pet30-His.sub.6-SUMO-AEY-Ssp.sup.N (P35N, E36N, R38N, V39I) (SEQ ID NO: 374) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSFGTEILTVEYGPLPIGKIVSEEINCSVY SVDNNGNIYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHRFLTTDYQLL AIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK
(88) The four batch mutants (Batches 5-8) and A136S point mutant on the Ssp.sup.C intein were cloned by inverse PCR using Pfu Ultra II HS Polymerase (Agilent) using plasmid 3 as a template and code the protein sequences shown below:
(89) TABLE-US-00028 Plasmid 15: Batch 5: pTXB1-Ssp.sup.C-MxeGyrA-His.sub.6 (V103I, V105I, I106A, G107T) (SEQ ID NO: 375) MIKIATRRSLGVQRIFDIGLPQDHNFLLANGAIAANCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH Plasmid 16: Batch 6: pTXB1-Ssp.sup.C-MxeGyrA-His.sub.6 (R115N, I116V, F117Y) (SEQ ID NO: 376) MVKVIGRRSLGVQNVYDIGLPQDHNFLLANGAIAANCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH Plasmid 17: Batch 7 pTXB1-Ssp.sup.C-MxeGyrA-His.sub.6 (L121V, P122E, Q123R) (SEQ ID NO: 377) MVKVIGRRSLGVQRIFDIGVERDHNFLLANGAIAANCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH Plasmid 18: Batch 8: pTXB1-Ssp.sup.C-MxeGyrA-His.sub.6 (L128A, A130K, A133F) (SEQ ID NO: 378) MVKVIGRRSLGVQRIFDIGLPQDHNFALKNGFIAANCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH Plasmid 19: SspC A136S: pTXB1-Ssp.sup.C-MxeGyrA-His.sub.6 (A136S) (SEQ ID NO: 379) MVKVIGRRSLGVQRIFDIGLPQDHNFLLANGAIASNCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH
(90) The gene for the fused Consensus DnaE sequence was codon-optimized for E. coli expression through IDT DNA and purchased as a gBlock. The DNA gBlock sequence is shown below:
(91) TABLE-US-00029 (SEQ ID NO: 380) TGCCTGTCTTACGACACAGAGATTCTGACCGTTGAATATGGATTCCTTCCT ATCGGTAAGATCGTGGAGGAACGGATTGAATGCACAGTCTATACGGTAGAT AAAAATGGCTTTGTGTATACACAACCTATTGCTCAGTGGCATAACCGGGGA GAACAGGAAGTTTTCGAATACTGCTTAGAAGACGGTTCGATTATCCGTGCA ACGAAAGATCACAAATTTATGACGACCGACGGTCAGATGTTACCGATTGAT GAGATTTTCGAACGGGGGTTAGACCTGAAACAAGTTGATGGTTTGCCGATG GTCAAGATCATTAGTCGTAAGAGTCTGGGCACTCAAAACGTCTACGATATT GGAGTAGAAAAAGATCATAATTTTTTGCTGAAGAATGGGCTGGTGGCCTCT AAC
(92) The expression plasmid for Cfa.sup.N was cloned using Gibson assembly into plasmid 1, yielding a vector coding for the following protein shown below:
(93) TABLE-US-00030 Plasmid 20: Cfa.sup.N: pET30-His.sub.6-SUMO-AEY-Cfa.sup.N (SEQ ID NO: 381) MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSD GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDL DMEDNDIIEAHREQIGGAEYCLSYDTEILTVEYGFLPIGKIVEERIECTVY TVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQML PIDEIFERGLDLKQVDGLP
(94) The expression plasmid for the Consensus C-intein was cloned using Gibson Assembly into plasmid 3, yielding a vector coding for the following gene:
(95) TABLE-US-00031 Plasmid 21: Cfa.sup.C: pTXB1-Cfa.sup.C-MxeGyrA-H6 (SEQ ID NO: 382) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNCITGDALVALPEGES VRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEG LRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGF ARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVAS VTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH
(96) Cfa constructs used for E. coli growth screen.
(97) Cfa plasmids used to screen the dependency of splicing at the +2 position of the C-extein were generating using restriction cloning into a previously generated plasmid.sup.2 containing a dual expression system of the split aminoglycoside phosphotransferase (Kan.sup.R) gene. The Cfa dual expression construct is shown below:
(98) Plasmids 22-25
(99) [KanR promoter]-[RBS]-[KanR.sup.N]-[Cfa.sup.N]-[iRBS]-[Cfa.sup.C-[CXN-KanR.sup.C]
(100) Following the promoter sequence, there are two separate E. coli ribosomal binding sites in this vector (RBS and iRBS). Each RBS is followed by one half of the split KanR-Intein construct, whose protein sequences are shown below (the Cfa intein is highlighted in bold).
(101) TABLE-US-00032 KanR.sup.N-Cfa.sup.N: (SEQ ID NO: 384) MEQKLISEEDLSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIY RLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPD DAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNS DRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFCLSYDT EILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFE YCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP Cfa.sup.C-KanR.sup.C (SEQ ID NO: 385) MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNCXNSVVTHGDFSLDN LIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGID NPDMNKLQFHLMLDEFF
(102) The +2 position of the C-extein is underlined, and is either phenylalanine, glycine, arginine, or glutamate.
(103) αDEC205-HC-Cfa.sup.N
(104) pCMV Plasmids containing the αDEC205 antibody light chain (LC), heavy chain (HC), and HC-intein fusions (HC-Npu.sup.N, HC-Mcht.sup.N, HC-Ava.sup.N) were obtained as previously described..sup.3 A codon-optimized Cfa DnaE sequence for mammalian cell expression was generated using JCAT.sup.4 and purchased as a gBlock through IDT DNA. The sequence is shown below:
(105) TABLE-US-00033 (SEQ ID NO: 386) TGCCTGAGCTACGACACCGAGATCCTGACCGTGGAGTACGGCTTCCTGCCC ATCGGCAAGATCGTGGAGGAGCGCATCGAGTGCACCGTGTACACCGTGGAC AAGAACGGCTTCGTGTACACCCAGCCCATCGCCCAGTGGCACAACCGCGGC GAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCATCATCCGCGCC ACCAAGGACCACAAGTTCATGACCACCGACGGCCAGATGCTGCCCATCGAC GAGATCTTCGAGCGCGGCCTGGACCTGAAGCAGGTGGACGGCCTGCCCGTG AAGATCATCAGCCGCAAGAGCCTGGGCACCCAGAACGTGTACGACATCGGC GTGGAGAAGGACCACAACTTCCTGCTGAAGAACGGCCTGGTGGCCAGCAAC
(106) The mammalian codon-optimized Cfa.sup.N sequence was then cloned into the pCMV HC-Npu.sup.N plasmid using restriction cloning to give a sequence coding for the following protein:
(107) TABLE-US-00034 Plasmid 26: HC-Cfa.sup.N: pCMV-HC-Cfa.sup.N (SEQ ID NO: 387) MGWSCIILFLVATATGVHSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDF YMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYL QMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSATTKGPSVYP LAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDL YTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVP EVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTIS KTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPA ENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEK SLSHSPGKASGGCLSYDTEILTVEYGELPIGKIVEERIECTVYTVDKNGFV YTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFER GLDLKQVDGLPGHHHHHHG
(108) Cfa.sup.C Intein for Ligation of Dendrimer:
(109) A plasmid containing the Cfa C-intein with a C-extein linker was cloned by inverse PCR into plasmid 21 and codes for the protein sequence shown below:
(110) TABLE-US-00035 Plasmid 27: Cfa.sup.C-link: pTXB1-H6-Cfa.sup.C-CFNSGG-MxeGyrA-H6 (SEQ ID NO: 388) MGHHHHHHSGVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNCFNSGG CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLF HSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDY AVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIA DELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHAHHHHHH
(111) The expression and purification protocols of all His.sub.6-SUMO-AEY-Int.sup.N (plasmids 1, 2, 5-14, 20) and Int.sup.C-GyrA-His.sub.6 (plasmids 3, 4, 15-19, 21 27) constructs were adapted from previously described methods..sup.1
(112) Expression of all His.sub.6-SUMO-AEY-Int.sup.N Constructs
(113) E. coli BL21(DE3) cells were transformed with an N-intein plasmid and grown at 37° C. in 1 L of LB containing 50 μg/mL of kanamycin. Once the culture had reached an OD.sub.600=0.6, 0.5 mM IPTG was added to induce expression (0.5 mM final concentration, 3 hr at 37° C.). The cells were pelleted via centrifugation (10,500 rcf, 30 min) and stored at −80° C.
(114) Purification of all His.sub.6-SUMO-AEY-Int.sup.N Constructs
(115) Purification of N-Intein Constructs for Batch Mutagenesis
(116) The cell pellets (from expression of plasmids 1, 2, 5-14) were resuspended in 30 mL of lysis buffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, pH 8.0) containing Roche Complete protease inhibitor cocktail. The resuspended cells were then lysed by sonication on ice (35% amplitude, 8×20 second pulses on/30 seconds off). The insoluble inclusion body containing the N-intein was recovered by centrifugation (35,000 rcf, 30 min). The supernatant was discarded and the pellet was resuspended in 30 mL of Triton wash buffer (lysis buffer with 0.1% triton X-100) and incubated at room temperature for 30 minutes. The Triton wash was then centrifuged at 35,000 rcf for 30 minutes. The supernatant was discarded, the inclusion body pellet was resuspended in 30 mL of lysis buffer containing 6M Urea, and the suspension was incubated overnight at 4° C. to extract and resolubilize the protein. This mixture was then centrifuged at 35,000 rcf for 30 minutes.
(117) The supernatant was then mixed with 4 mL of Ni-NTA resin (for affinity purification using the His.sub.6 tag) and incubated at 4° C. for 30 minutes to batch bind the protein. This mixture was loaded on a fritted column, the flow through was collected, and the column was washed with 5 column volumes (CV) of lysis buffer with 6M Urea and 5 CV of lysis buffer with 25 mM imidazole and 6M urea. The protein was then eluted in four 1.5 CV fractions of lysis buffer with 250 mM imidazole and 6M Urea. The first two elution fractions were generally found by SDS-PAGE (12% Bis-Tris gel, run for 50 minutes at 170V) to contain the expressed protein and were combined for refolding.
(118) The N-inteins were refolded by stepwise dialysis into lysis buffer with 0.5 mM DTT at 4° C. This refolded protein was then treated with 10 mM TCEP and Ulp1 protease (overnight, RT) to cleave the His.sub.6-SUMO expression tag. The solution was then mixed with 4 mL Ni-NTA resin and incubated for 30 minutes at 4° C. The slurry was applied to a fritted column and the flow through was collected together with a 3 CV wash with lysis buffer. The protein was then treated with 10 mM TCEP, concentrated to 10 mL, and further purified by size exclusion chromatography using an S75 16/60 gel filtration column employing degassed splicing buffer (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2) as the mobile phase. Fractions were analyzed by SDS-PAGE, analytical RP-HPLC, and ESI-MS. Pure protein was stored by flash-freezing in liquid N.sub.2 following the addition of glycerol (20% v/v). Note: during the refolding step, significant protein precipitation was observed for Batch 3, suggesting it is prone to aggregation.
(119) Purification of Cfa.sup.N:
(120) The cell pellet (from expression of plasmid 20) was first resuspended in 30 mL of lysis buffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, pH 8.0) containing the Roche Complete protease inhibitor cocktail. The cells were then lysed by sonication (35% amplitude, 8×20 second pulses on/30 seconds off), and the lysate was pelleted by centrifugation (35,000 rcf, 30 min). The supernatant was incubated with 4 mL of Ni-NTA resin for 30 minutes at 4° C. to enrich for the soluble Cfa.sup.N protein. The slurry was then loaded onto a fritted column, and the column was washed with 20 mL of wash buffer 1 (lysis buffer) followed by 20 mL of wash buffer 2 (lysis buffer with 25 mM imidazole). Finally, the protein was eluted from the column with 4×1.5 CV of elution buffer (lysis buffer+250 mM imidazole).
(121) The desired protein, which was present in elution fractions 1 and 2 as determined by SDS-PAGE (12% bis-tris gel run in MES-SDS running buffer at 170V for 50 minutes), was then dialyzed into lysis buffer for 4 hours at 4° C. Following dialysis, the protein was treated with 10 mM TCEP and Ulp1 protease overnight at room temperature to cleave the His.sub.6-SUMO expression tag. The solution was then incubated with 4 mL Ni-NTA resin for 30 minutes at 4° C. The slurry was applied to a fritted column and the flow through was collected together with a 3 CV wash with lysis buffer. The protein was then treated with 10 mM TCEP, concentrated to 10 mL, and purified over an S75 16/60 gel filtration column employing degassed splicing buffer (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2) as the mobile phase. Fractions were analyzed by SDS-PAGE (12% bis-tris gel run in MES-SDS running buffer at 170V for 60 minutes), analytical RP-HPLC, and ESI-MS. Pure Protein was stored in glycerol (20% v/v) and flash-frozen in liquid N.sub.2.
(122) Semisynthesis of Int.sup.C-CFN Constructs
(123) E Coli BL21 (DE3) cells were transformed with the appropriate pTXB1-Int.sup.C-GyrA-H.sub.6 plasmid (plasmids 3, 4, 15-19, 21) and grown in 2 L of LB media containing ampicillin (100 μg/mL) at 37° C. Once the culture had reached an OD.sub.600=0.6, expression was induced by the addition of IPTG (0.5 mM, 3 hours, 37° C.). Cell pellets were harvested by centrifugation (10,500 rcf, 30 min), resuspended in lysis buffer, and lysed by sonication on ice (35% amplitude, 10×20 second pulses on/30 seconds off). The protein in the soluble fraction was isolated by centrifugation (35,000 rcf, 30 min) and then enriched by Ni-NTA purification (4 mL beads, carried out as described for N-intein constructs). Following elution in lysis buffer with 250 mM imidazole, the imidazole was removed by dialysis into fresh lysis buffer. The ligation was then carried out overnight at room temperature with the addition of 10 mM TCEP, the Roche Complete protease inhibitor cocktail, 100 mM MESNa, 5 mM EDTA, and 5 mM CFN-NH.sub.2 (pH 7.0). The ligated Int.sup.C-CFN peptide was acidified with 0.5% TFA and purified via RP-HPLC on a C.sub.18 preparative column: Gradient=10% B for 10 minutes (isocratic) followed by 20-60% B over 60 minutes. The purity of each protein was determined by analytical RP-HPLC and its identity was confirmed by ESI-MS.
(124) Isolation of Cfa.sup.C-Link-MESNa
(125) The Cfa.sup.C-link-MESNa peptide used for the semisynthesis of the Intein-dendrimer fusion was expressed and purified exactly as described above for the Int.sup.C-CFN constructs (expression from plasmid 27). However, no tripeptide was added during the final ligation step, instead resulting in thiolysis of the intein and formation of an α-thioester. This Cfa.sup.C-MESNa α-thioester was then purified by preparative RP-HPLC. Fractions were analyzed by ESI-MS, combined, and lyophilized.
(126) Analysis of protein trans-splicing by RP-HPLC and ESI-MS for Batch Mutants.
(127) Splicing reactions were carried out as adapted from a previously described protocol. Briefly, N- and C-inteins (15 μM Int.sup.N, 10 μM Int.sup.C) were individually preincubated in splicing buffer (100 mM sodium phosphates, 150 mM NaCl, 1 mM EDTA, pH 7.2) with 2 mM TCEP for 15 minutes. All splicing reactions were carried out at 30° C. unless otherwise indicated. Splicing reactions comparing the tolerance of Npu and Cfa to chaotropic agents were carried out with the indicated concentration of either Urea or guanidine hydrochloride. Splicing was initiated by mixing equal volumes of N- and C-inteins with aliquots removed at the indicated times and quenched by the addition of 8M guanidine hydrochloride, 4% TFA (3:1 v/v). For all splicing reactions containing either Npu.sup.C-CFN or Cfa.sup.C-CFN, reaction progress was monitored by RP-HPLC. For all splicing reactions containing Ssp.sup.C-CFN, reaction progress was monitored by ESI-MS (samples desalted with ZipTip prior to injection) due to poor chromatographic resolution of each state as seen previously..sup.1 Splicing for Batch 3 and for Cfa at 80° C. (15 minute preincubation) were both observed to be inefficient, reaching ˜50% completion. This is likely due to aggregation (and inactivation) of the N-intein. Note, shorter preincubations of Cfa at 80° C. led to more efficient splicing.
(128) Kinetic Analysis of Trans-Splicing Reactions of Batch Mutants:
(129) Kinetic analysis was carried out as previously described..sup.1 Briefly, five species (1-5) are separated by RP-HPLC, and peak areas are determined. For ESI-MS, peak areas are calculated for species 1-4. Each individual peak was normalized against the total area of all peaks combined and reaction progress curves were plotted (n=3). The data were then fit in ProFit to the analytical solution to the coupled differential rate equation for the three state kinetic splicing model. Because the starting material cannot be separated from the linear thioester using this assay, the three state kinetic model collapses the binding step and the first two steps of the splicing reaction into one equilibrium. Each splicing reaction was carried out in triplicate with each replicate analyzed separately. The mean and standard deviation for all values (n=3) are reported.
(130) Kinetic Analysis of Overall Trans-Splicing Reactions for Npu and Cfa
(131) All splicing reactions comparing Npu and Cfa were separated by RP-HPLC with peak areas once again calculated using the manufacturer's software. For these reactions, peak areas for the starting material and branched intermediate (species 1 and 2) and product (species 3, 4, 5) were calculated. The data was then fit to the first order rate equation using the GraphPad Prism software.
[P](t)=[P]max.Math.(1−e.sup.−kt)
(132) Where [P] is the normalized intensity of product, [P].sub.max is this value at t=∞ (the reaction plateau), and k is the rate constant (s.sup.−1). The mean and standard deviation (n=3) are reported.
(133) Generation and refinement of the DnaE Intein Multiple Sequence alignment.
(134) Homologues of Npu DnaE were identified through a BLAST.sup.5 search of the NCBI.sup.6 (nucleotide collection) and JGI.sup.7 databases using the Npu DnaE protein sequences. This led to the identification of 105 proteins with >60% sequence identity. For N-inteins with long C-terminal tails, the proteins were truncated to 102 residues, the length of Npu. For N-inteins from the JGI database, the point of truncation was determined by the results of the BLAST program (the last residue identified in the Blast search was selected as the truncation point). Next, a multiple sequence alignment (MSA) was generated of the fused sequence (i.e. the N-intein connected to the C-intein) of all 105 inteins in Jalview (
(135)
(136) E. coli Kan.sup.R screen for Cfa extein dependency.
(137) The protein splicing coupled kanamycin resistance (Kan.sup.R) assay was carried out as previously described..sup.2,9 Briefly, a plasmid coding for a fragmented aminoglycoside phosphotransferase fused to a split intein (Cfa) with either F, G, R, or E present at the +2 position of the C-extein (plasmids 22-25) was transformed into DH5α competent cells and grown in starter cultures overnight (LB Broth, 100 μg/mL ampicillin, 18 hrs). These cultures were then diluted twenty-fold into a 96 well plate, and E. coli growth was measured at various concentrations of kanamycin (2.5, 10, 25, 50, 100, 250, 1000 μg/mL kanamycin with 100 μg/mL ampicillin). The cell optical density at 650 nm (OD.sub.650) at the 24-hour end point was fit to a dose response curve with variable slope.
(138)
(139) Where OD.sub.min was fixed to background absorbance at 650 nm. Each assay was carried out in triplicate, fit separately, and IC.sub.50 values are reported as the mean and standard deviation of IC.sub.50 for these three separate measurements.
(140) Protein Trans Splicing of Extracted Inclusion Body
(141) E. coli inclusion bodies containing His.sub.6-Sumo-Cfa.sup.N expression (plasmid 20) were resuspended and extracted overnight at 4° C. in lysis buffer containing 6M urea. Following centrifugation (35,000 rcf, 30 min), the supernatant was removed and the protein enriched by Ni-NTA under denaturing conditions (as described above). However, instead of refolding the protein, trans-splicing was directly initiated by the addition of Cfa.sup.C-CFN (10 μM Cfa.sup.C, 2 mM TCEP, 2 mM EDTA, 2 hrs, RT). Reaction progress was monitored by SDS-PAGE.
(142) αDec205-HC-Int.sup.N Test Expression and Splicing
(143) Test Expression of HC-Npu.sup.N, HC-Mcht.sup.N, HC-Ava.sup.N, HC-Cfa.sup.N
(144) Expression of all mAb constructs was carried out as previously described..sup.3 Briefly, plasmids encoding the αDec205-LC and the αDec205-HC-Int.sup.N were co-transfected into HEK293T cells and incubated for 96 hr (5% CO.sub.2). The cells were spun down (5 minutes, 1,000 rcf), 15 μL of media for each intein fusion was mixed with 5 μL of 4× loading dye, and run on a 12% Bis-Tris gel in MES-SDS running buffer (170V for 50 minutes). The protein was then analyzed by western blot (transferred to a PVDF membrane, blotting against αMouse IgG). Expression yield was measured as the amount of HC-Int.sup.N in the media as determined by densitometry. To account for varying cell growth and survival, the yield was normalized using an α-actin blot of the HEK293T cell lysate (5 s sonication, 35% amplitude, in 1× loading dye) and then represented relative to the expression of HC-Cfa.sup.N. Four replicates of this test expression were carried out, and the mean was calculated with error represented as the standard deviation.
(145) Protein Trans-Splicing in Growth Media
(146) Following the 96 hr expression at 37° C. of the mAB-Ava.sup.N and mAB-Cfa.sup.N constructs described above, the media was spun down (1,000 rcf, 5 minutes). The supernatant was then mixed with the Cfa.sup.C-CFN peptide (semisynthesis of expressed plasmid 21) and incubated for 2 hours at room temperature (1 μM Cfa.sup.C-CFN, 2 mM TCEP, 2 mM EDTA). The splicing reactions were analyzed by SDS-PAGE (12% Bis-Tris run in MES-SDS running buffer at 170V for 50 minutes) followed by western blot (αMouse IgG).
(147) Peptide and Dendrimer Synthesis
(148) Cys-Gly-Lys(Fluorescein). This peptide was synthesized by manual addition of reagents on the Rink Amide resin according to a previously published procedure..sup.2
(149) ##STR00006## ##STR00007## ##STR00008##
(150) Compound 2 (dendrimer thioester). This compound was synthesized on the solid phase using the route outlined in Supplemental Scheme 1 on a scale of 400 mg of Rink Amide resin (substitution: 0.47 mmol/g, 188 μmol). General procedures are given first, followed by any specific methods for this peptide. The Fmoc group was removed with 3 mL of 20% piperidine in DMF and performed twice (one deprotection for 30 sec followed by an additional deprotection for 15 min). After each deprotection step, as well as all subsequent synthetic steps, flow washes were used (3×5 sec. with ˜5 mL of DMF each). Coupling was performed using 4 eq. of monomer, 4 eq. of either HBTU and 8 eq. of DIPEA with no pre-activation unless otherwise stated. Double couplings were used for all residues to ensure complete acylation.
(151) The Trityl protecting group was selectively removed using 1% TFA, 5% TIS in DCM using a total of 30 mL (10×3 mL) of deprotection cocktail. Thorough washing of the resin with DCM both during and after these cycles ensured the removal of any liberated Trityl species. The resin was also neutralized with 5% DIPEA in DMF before the next coupling was undertaken. The Alloc group was deprotected using 0.1 eq of tetrakis(triphenylphosphine) palladium(0), 20 eq of phenylsilane in DCM for 3×45 min each. Thorough washing of the resin with DCM during and after these cycles was used, as well as a 5% DIPEA in DMF wash before the next coupling. The glutaric anhydride monomer was used as a preactivated dicarboxylic acid to allow the formation of the thioesters (i.e. to have a free resin-bound carboxylic acid to functionalize). 20 eq of glutaric anhydride and 10 eq of DIPEA (relative to the number of amines to be acylated) was added to the resin and allowed to react for one hour. The resin was then washed and the coupling was repeated to ensure complete reaction of the resin bound primary amines. To form the resin bound thioesters, 30 eq of methyl thioglycolate, 5 eq of PyAOP and 10 eq of DIPEA (relative to the number of carboxylates) in DMF was added to the resin and allowed to react for one hour. The resin was washed with excess DMF and the coupling procedure was repeated an additional two times.
(152) Cleavage was performed with 95% TFA, 2.5% TIS and 2.5% H.sub.2O for two hours at room temperature. The peptide was then precipitated with diethyl ether, dissolved in water with 0.1% TFA and analyzed via RP-HPLC. The crude material was purified via semi-preparative scale RP-HPLC, and the desired fractions were analyzed, pooled and lyophilized. RP-HPLC characterization: gradient 0-73% B, t.sub.r=18.4 min. Expected Mass: 2198.86 Da. Found: 2198.82 Da.
(153) Compound 3 (dendrimer fluorescein).
(154) Compound 3 was synthesized by native chemical ligation (scheme 2). Compound 2 was dissolved in ligation buffer and mixed with five eq. of Cys-Gly-Lys(Fluorescein) (1 mM 2, 5 mM peptide, 4M Guanidine, 100 mM phosphate, 150 mM NaCl, 100 mM MPAA, 20 mM TCEP, pH 7.0) and allowed to react overnight at room temperature. Deprotection of the thiazolidine was then accomplished by the addition of 0.1M methoxyamine (final concentration) and decreasing the pH of the ligation buffer to 4.0 (overnight, RT).
(155) When attempting to purify compound 3 by RP-HPLC, we noticed that it displayed poor solubility when acidified and diluted in water. However, Cys-Gly-Lys(Fluorescein), MPAA, and methoxyamine all remained in solution. From this observation, we purified 3 by selective precipitation following 10-fold dilution in water with 0.1% TFA. The precipitated powder was isolated by centrifugation (17,000 rcf, 5 min), and then redissolved (100 mM phosphate, 150 mM NaCl, pH 7.2) to wash away any remaining contaminants. Once again, the solution was precipitated by acidification and isolated by centrifugation (17,000 rcf, 5 min). This isolated powder was then lyophilized. Expected mass: 4417.8 Da. Found: 4417.5 Da.
(156) ##STR00009## ##STR00010## ##STR00011##
(157) Compound 1: (Cfa.sup.C-Dendrimer)
(158) Compound 1 was synthesized by expressed protein ligation. Compound 3 was dissolved in ligation buffer and mixed with 1.5 eq of the Cfa.sup.C-MESNa thioester (100 μM 3, 150 μM Cfa.sup.C-MESNa, 4M Guanidine, 100 mM phosphate, 150 mM NaCl, 20 mM TCEP, 100 mM MPAA). The reaction was allowed to proceed overnight at room temperature. The ligated product was then purified by semi-preparative RP-HPLC. Desired fractions were pooled and lyophilized. Expected mass: 9860.8 Da. Found: 9860.3 Da.
(159) Protein Trans-Splicing of dendrimer with αDec205 mAb.
(160) The αDec205 mAb with Cfa.sup.N fused to its C-terminus was expressed as described above. Following the 96 hr expression, the media was concentrated 10-fold in an Amicon 30K concentrator (0.5 mL). Compound 1 was dissolved in splicing buffer (100 mM phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2) and then mixed with the concentrated media (2 μM compound 1, 2 mM TCEP, 1 mM EDTA) and the reaction allowed to proceed for 2 hrs at room temperature. The splicing mixture was then analyzed by SDS-PAGE (12% Bis-Tris run in MES-SDS running buffer at 170V for 50 minutes) and imaged on a fluorescence imager. This was followed by transfer to a PVDF membrane and western blot analysis (αMouse IgG).
(161) The invention allows for the formation of various complexes between a split intein fragment and a compound. Several such complexes and compounds are illustrated in the table of
Example 2
(162) A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. In addition to a mandatory catalytic Cys, Ser, or Thr residue at position +1 (i.e., the first residue within the C-extein), there is a bias for residues resembling the proximal N- and C-extein sequence found in the native insertion site. Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of PTS-based methods..sup.23, 24 Accordingly, there is a need for split inteins whose activities are minimally affected by local sequence environment. For DnaE inteins, extein sequence preferences are largely confined to the catalytic cysteine at the +1 position and large hydrophobic residues that are preferred at the +2 position..sup.25
(163) In this example, a “EKD” to “GEP” loop mutation into residues 122-124 of Cfa (Cfa.sub.GEP) was engineered and resulted in increased promiscuity at the +2 position of the C-extein in a kanamycin resistance assay (
(164) The following sequences represent the engineered inteins:
(165) The Cfa C-intein with the “GEP” mutation that imparts more “promiscuous” activity according to an embodiment of the invention is:
(166) TABLE-US-00036 (SEQ ID NO: 389) VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.
(167) An example of a fusion intein of the Cfa N-intein and Cfa C-intein with the “GEP” mutation of SEQ ID: 389) is:
(168) TABLE-US-00037 (SEQ ID NO: 390) CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRG EQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLPV KIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.
(169)
(170) Furthermore, this same tolerance for varying extein sequences was also observed in the cyclization of eGFP in E. coli (
(171)
(172) The following claims are thus to be understood to include what is specifically illustrated and described above, what is conceptually equivalent, what can be obviously substituted and also what essentially incorporates the essential idea of the invention. Those skilled in the art will appreciate that various adaptations and modifications of the just-described preferred embodiment can be configured without departing from the scope of the invention. The illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.
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