METHOD FOR SCREENING PORE-FORMING MEMBRANE PROTEINS, MEMBRANE TRANSPORTERS AND MOLECULAR SWITCHES
20230050056 · 2023-02-16
Inventors
Cpc classification
G01N33/6872
PHYSICS
International classification
Abstract
The present invention relates to a method for screening pore-forming membrane proteins, membrane transporters and molecular switches. The invention also relates to a kit for carrying out the method.
Claims
1. A screening method comprising the steps of: a) providing a cell and a medium surrounding the cell, wherein the cell has a cell membrane impermeable to a reporter substance, wherein the quotient of the concentration of the reporter substance in the interior of the cell and the concentration of the reporter substance in the medium surrounding the cell is at least 2 or at most 0.5; b) detecting a signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of a sensor protein expressed in the cell; c) inducing the expression of a membrane protein that increases the permeability of the cell membrane to the reporter substance; d) detecting the signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of the sensor protein expressed in the cell; e) calculating one or more comparative parameters from the signal strengths detected in steps b) and d), wherein step b) is carried out before step c) and wherein step d) is carried out after step c), wherein steps a) to e) are carried out for at least two membrane proteins having different amino acid sequences and/or for at least two sensor proteins having different amino acid sequences.
2. The screening method according to claim 1, comprising the further step of: f) determining the DNA sequence encoding the membrane protein and/or the DNA sequence encoding the sensor protein.
3. The screening method according to claim 1, wherein the cell is a microorganism.
4. The screening method according to claim 1, wherein the cell is Escherichia coli, and the cell membrane is the inner membrane of E. coli.
5. The screening method according to claim 2, wherein steps a) to f) are carried out for at least 5 membrane proteins with different amino acid sequences.
6. The screening method according to at least claim 2, wherein steps a) to f) are carried out for at least 5 sensor proteins with different amino acid sequences.
7. The screening method according to claim 1, wherein the signal is selected from one or more of a fluorescence signal, a bioluminescence signal, and an absorption signal.
8. The screening method according to claim 1, wherein the sensor protein is selected from the group of Ca.sup.2+-specific protein sensors and L-Glu-specific protein sensors.
9. The screening method according to claim 1, wherein the reporter substance is selected from the group consisting of cations and amino acids.
10. The screening method according to claim 1, wherein the membrane protein is selected from the group consisting of pore-forming membrane proteins and membrane transporters.
11. The screening method according to claim 1, wherein the membrane protein is selected from the group consisting of holins, pinholins, and ion channels.
12. The screening method according to claim 1, wherein the membrane protein is selected from the group consisting of S.sup.21-68 (SEQ ID NO:1), S.sup.21-71 (SEQ ID NO:2), S.sup.21-71 M4A (SEQ ID NO:3), .sup.SGSΔTMD1S.sup.2168 (SEQ ID NO:4), .sup.TVMVΔTMD1-S.sup.2168 (SEQ ID NO:5), S.sup.105 (SEQ ID NO:6), S.sup.107 (SEQ ID NO:7), S.sup.107-M3A (SEQ ID NO:8), T4 pinholin (SEQ ID NO:9), T4.sup.ΔC-Tail (SEQ ID NO:10), .sup.ΔN-TailT4.sup.ΔC-Tail (SEQ ID NO:11), K.sub.CV.sup.NTS (SEQ ID NO: 2), K.sub.CV.sup.NTS G77S (SEQ ID NO:13), K.sub.CV.sup.PBCV1 (SEQ ID NO:14), HokB (SEQ ID NO:15), TisB (SEQ ID NO:16), αHLA (SEQ ID NO:17), cWZA (SEQ ID NO:18), BM2 (SEQ ID NO:19), HCV TME1 (SEQ ID NQ:20), HCV TME2 (SEQ ID NO:21) and variants thereof having sequence identity to at least one of the sequences of SEQ ID NO: 1-21 of at least 90%.
13. The screening method according to claim 1, wherein step d) is carried out at most 120 minutes after step c).
14. The screening method according to claim 1, wherein step d) is carried out at least five times, and the calculation of the comparative parameter from the signal strengths according to step e) includes empirically fitting the data obtained on the signal strengths with a sigmoid function, wherein the comparative parameter calculated from the signal strengths is the half-maximum time c and/or the maximum slope d.
15. The screening method according to claim 14, wherein the data obtained on the signal strengths are fitted empirically with the following equation 1
16. The screening method according to claim 1, wherein the cell is a prokaryotic microorganism.
17. A kit for carrying out the screening method according to claim 1, the kit comprising: i. DNA encoding at least two membrane proteins and DNA encoding a sensor protein, or ii. DNA encoding at least two sensor proteins and DNA encoding a membrane protein.
18. The screening method according to claim 2, wherein steps a) to f) are carried out for at least 20 membrane proteins with different amino acid sequences.
19. The screening method according to claim 2, wherein steps a) to f) are carried out for at least 50 membrane proteins with different amino acid sequences.
20. The screening method according to claim 2, wherein steps a) to f) are carried out for at least 10.sup.2 to 10.sup.8 membrane proteins with different amino acid sequences.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0071]
[0072]
[0073]
[0074]
[0075]
[0076]
[0077]
[0078]
[0079]
[0080]
[0081]
[0082]
[0083]
[0084]
[0085]
[0086]
[0087]
[0088]
[0089]
[0090]
[0091]
[0092]
[0093]
[0094]
[0095]
[0096]
[0097]
[0098]
[0099]
EXAMPLES
[0100] The present invention is to be explained in more detail by the following examples.
Overview of Expression Constructs and Screening Strains for Nanopores and Ion Channels
Protein of Expression Constructs:
[0101] pPRO24: The pPRO24 plasmid contains a class-A on and a titrable propionate-inducible promoter (Environmental Microbiology 2005, 71, 6856-62). This enables flexible control of the expression of a molecular sensor, such as the Ca.sup.2+-specific R- and G-GECO (Science, 2011, 333, 1888-1891).
[0102] pOSIP-CH: The pOSIP-CH enables the incorporation of gene expression cassettes of co-expression into defined sites of the E. coli genome. Specifically, the incorporation is made possible by co-expression of the HK022 integrase. 25 Integration of gene expression cassette, e.g., for the R and G-GECO sensors, allows more stable propagation and expression of the R- and G-GECO cassettes in E. coli (caused by uniform copy numbers).
[0103] pACYCT2: The pACYCT2 plasmid contains a class-B on (p15A)+chloramphenicol resistance and a tac promoter induced by IPTG (Nucleic Acids Research, 2013, 41, e150). Used as expression vector for different pinholins and variants, derived therefrom, of the S.sup.21-68 pinholins: In particular, the pore-forming and non-pore-forming membrane peptides .sup.SGSΔTMD1-S.sup.2168 (PNAS, 2009, 106, 18966-18971) and .sup.TVMVΔTMD1-S.sup.21-68.
[0104] pTeT7W: Dual promoter vector based on the pACYCT2 backbone with p15A ori+chloramphenicol resistance and lacI repressor for independent co-expression of two proteins. In addition to the given elements of the pACYCT2, a TetR expression cassette together with promoter from the pASK and the tac was replaced with the T7 promoter from the pET24. This allows the expression of the two proteins by means of IPTG and tetracycline.
[0105] pCTRL2: Expression vector for the expression of toxic membrane channels and membrane peptides. Based on the pACYCT2 vector. The gene expression cassette is flanked on both sides by transcription terminators (ACS Synthetic Biology, 2015, 20, 4, 265-73) and the basal expression of potentially non-specific proteins is to be suppressed. The expression is carried out under control of a lacI repressor mutant with increased suppression of gene expression (Microbial Cell Factories, 2013, 12, 67). The underlying promoter is denoted in the present disclosure by T7.wt.sRBS in each case. The transcription cassette in the context of pCTRL2 with T7.wt.sRBS promoter is shown in SEQ ID NO:50 by way of example for the expression of the fluorescent protein mkO-kappa. The fluorescent protein mkO-kappa is in each case replaced by a membrane protein in the individual experiments.
[0106] pCTRL2.T7.100.sRBS: Expression vector for the expression of toxic membrane channels and membrane peptides. Based on the pACYCT2 vector. The gene expression cassette is flanked on both sides by transcription terminators (ACS Synthetic Biology, 2015, 20, 4, 265-73) to support the basal expression of potentially non-specific proteins. The expression is carried out under control of a lacI repressor mutant with increased suppression of gene expression (Microbial Cell Factories, 2013, 12, 67). In comparison to pCTRL2, the distance in pCTRL2.T7.100.sRBS between the ribosome-binding site and the binding site for the lac repressor is shortened in this expression vector and thus causes a lower expression of the respective nanopore. The underlying promoter is denoted in the present disclosure by T7.100.sRBS in each case. The transcription cassette in the context of pCTRL2.T7.100.sRBS with T7.100.sRBS promoter is shown in SEQ ID NO:51 by way of example for the expression of the fluorescent protein mkO-kappa. The fluorescent protein mkO-kappa is in each case replaced by a membrane protein in the individual experiments.
E. coli Strains for Assay Development and Screening:
[0107] BL21(DE3) with pPRO24R-GECO1. The BL21(DE3) E. coli were made chemically competent together with pPRO24-R-GECO1 or pPRO24-G-GECO1. This strain enables the efficient transformation and screening of pore-forming membrane peptides, nanopores and ions channels that are expressed via pACYCT2, pTeT7W or pCTRL2.
[0108] BL21(DE3) with genomically Integrated R- and G-GECO1: This strain contains the expression cassette for R- or G-GECO1 in a genomically integrated variant. Specifically, the gene for R- or G-GECO1 was stably integrated by means of the pOSIP-CH plasmid into the attB site of the E. coli genome (ACS Synthetic Biology, 2013, 2, 537-41). This strain enables the efficient transformation and screening of pore-forming membrane peptides, nanopores and ions channels that are introduced via pACYC2, pTeT7Woder pCTRL2.
Overview of Fluorescent Protein Sensors, Nanopores and Ion Channels
Overview of Fluorescent Protein Sensors:
[0109] R-GECO-1: Red fluorescent Ca.sup.2+-specific protein sensor (Science, 2011, 333, 1888-1891). Is preferably used in the microtiter plate and in the colony format as genetically encoded reporter to detect the inflow of Ca.sup.2+ after the formation of a pore in the inner membrane of E. coli.
[0110] G-GECO-1: Green fluorescent Ca.sup.2+-specific protein sensor (Science, 2011, 333, 1888-1891). Is preferably used in the flow cytometer as genetically encoded reporter to detect the inflow of Ca.sup.2+ after the formation of a pore in the inner membrane of E. coli.
[0111] iGluSnFR: Denotes a class of fluorescent protein sensors specifically for L-Glu (Nature Methods, 2013, 10, 162-70). Used to detect low-molecular materials and substances beyond Ca.sup.2+ or to cause nanopore-dependent change in the fluorescent signal.
Overview of Membrane Proteins. Ion Channels, and Pore-Forming Membrane Peptides:
Holins and Pinholins:
[0112] S.sup.21-68: Wild-type of the S.sup.21-68 pinholin of bacteriophage P21 with pronounced and defined pore-forming properties (PNAS, 2009, 106, 18966-18971). Exact mechanism of pore formation is currently unclear. Presumed is first an inactive conformation in the form of an antiparallel a-helix with respectively cytosolic N- and C-termini, which collect in the inner membrane of the inner membrane of E. coli. A concentration-related increase of S.sup.21-68 subsequently results in flipping of the first transmembrane domain by the lipid bilayer and in pore formation. Presumed is a defined pore consisting of seven subunits and a membrane channel consisting of the second transmembrane domain, which is stabilized by homomeric interactions of the first transmembrane domain in the periplasma. (PNAS, 2013, 110, E2054-E2063)
[0113] Polypeptide Sequence:
TABLE-US-00001 (SEQ ID NO: 1) MDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGFL TYLTNLYFKIREDRRKAARGE
[0114] S.sup.21-68 truncations: N-terminal truncations of the wild-type of the S.sup.21-68 pinholin of bacteriophage P21.
[0115] The polypeptide sequences are summarized in the following table:
TABLE-US-00002 Trunc- ation Amino acid sequence S2166 MKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSL VLGFLTYLTNLYFKIREDRRKAARGE (SEQ ID NO: 28) S2164 MSTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLV LGFLTYLTNLYFKIREDRRKAARGE (SEQ ID NO: 29) S2162 MGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLG FLTYLTNLYFKIREDRRKAARGE (SEQ ID NQ: 30) S2160 MAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGFL TYLTNLYFKIREDRRKAARGE (SEQ ID NO: 31) S2158 MGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGFLTY LTNLYFKIREDRRKAARGE (SEQ ID NO: 32) S2156 MSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGFLTYLT NLYFKIREDRRKAARGE (SEQ ID NO: 33) S2154 MGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGFLTYLTNLY FKIREDRRKAARGE (SEQ ID NO: 34) S2152 MAGYWFLQWLDQVSPSQWAAIGVLGSLVLGFLTYLTNLYFK IREDRRKAARGE (SEQ ID NO: 35) S2150 MYWFLQWLDQVSPSQWAAIGVLGSLVLGFLTYLTNLYFKIR EDRRKAARGE (SEQ ID NO: 36) S2148 MFLQWLDQVSPSQWAAIGVLGSLVLGFLTYLTNLYFKIRED RRKAARGE (SEQ ID NO: 37) S2146 MQWLDQVSPSQWAAIGVLGSLVLGFLTYLTNLYFKIREDRR KAARGE (SEQ ID NO: 38) S2144 MLDQVSPSQWAAIGVLGSLVLGFLTYLTNLYFKIREDRRKA ARGE (SEQ ID NO: 39) S2142 MQVSPSQWAAIGVLGSLVLGFLTYLTNLYFKIREDRRKAAR GE (SEQ ID NQ: 40) S2140 MSPSQWAAIGVLGSLVLGFLTYLTNLYFKIREDRRKAARGE (SEQ ID NO: 41) S2138 MSQWAAIGVLGSLVLGFLTYLTNLYFKIREDRRKAARGE (SEQ ID NO: 42)
[0116] S.sup.21-71: Wild-type of the S.sup.21-71 anti-pinholin of bacteriophage P21. In comparison to S.sup.21-68, the N-terminus is expanded by three additional amino acids MKS, which limit the pore-forming properties. The exact mechanism as to how the pore formation is limited is still unclear according to current knowledge (Current Opinion in Microbiology, 2013, 16, 790-797). Presumed is a role of positive charges that prevent flipping of the first transmembrane domain.
[0117] Polypeptide Sequence:
TABLE-US-00003 (SEQ ID NO: 2) MKSMDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE
[0118] S.sup.21-71 M4A: Mutant of the S.sup.21-71 anti-pinholin in which the second methionine is mutated to alanine at the fourth position. Thus, in principle, prevents the expression of the wild-type S.sup.21-68 pinholin, which in the case of S.sup.21-71 can also be expressed in principle by moving the translation initiation site by three amino acids. In comparison to the S.sup.21-71 anti-pinholin, the pore formation is thus further delayed.
[0119] Polypeptide Sequence:
TABLE-US-00004 (SEQ ID NO: 3) MKSADKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE
[0120] N-terminal S.sup.21-71 mutants: N-terminal mutants of the S.sup.21-71 anti-pinholin with the following polypeptide sequence:
MXXXDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGFLT YLTNLYFKIREDRRKAARGE (SEQ ID NO:43), wherein A, R, N, D, Q, E, G, H, I, L, K, F, S, T, W, Y or V can be provided independently of one another at each of the positions indicated by “X.”
[0121] Particularly Preferred Sequences are:
TABLE-US-00005 Clone P14E5: (SEQ ID NO: 44) MYLEDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE Clone P14C11: (SEQ ID NO: 45) MNFEDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE Clone P15D3: (SEQ ID NO: 46) MDWDDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE Clone P7D10: (SEQ ID NO: 47) MAGWDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE Clone P3G3: (SEQ ID NO: 48) MWRGDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE Clone P3E5: (SEQ ID NO: 49) MFQWDKISTGIAYGTSAGSAGYWFLQWLDQVSPSQWAAIGVLGSLVLGF LTYLTNLYFKIREDRRKAARGE
[0122] .sup.SGSATMD1-S.sup.2168: Fragment of the S.sup.21-68 pinholin in which the first transmembrane domain ATMD1 was deleted. Expression is in principle toxic, presumably due to the ability to form at least transient pores in the inner membrane of E. coli (PNAS, 2009, 106, 18966-18971; Molecular Microbiology, 2010, 76, 68-77).
[0123] Polypeptide Sequence:
TABLE-US-00006 (SEQ ID NO: 4) MSGSMWAAIGVLGSLVLGFLTYLTNLYFKIREDRRKAARGE
[0124] .sup.TVMVΔTMD1-S.sup.2168: Fragment of the S.sup.21-68 pinholin in which the first transmembrane domain TMD1 is missing and the cutting sequence ETVRFQ'S precedes the TVMV protease N-terminal. Expression is non-toxic, presumably likely due to the proteolytic TVMV interface, which greatly limits the pore-forming properties.
[0125] Polypeptide Sequence:
TABLE-US-00007 (SEQ ID NO: 5) MSSSGGSETVRFQSGSMWAAIGVLGSLVLGFLTYLTNLYFKIREDRRKA ARGE
[0126] S.sup.105: Wild-type of the S.sup.105 pinholin of bacteriophage A with pronounced pore-forming properties (PNAS, 2010, 107, 2219-23). In contrast to the S.sup.21-68 pinholin, it is presumed that the S105 holin forms large, pm-large pores. However, the exact mechanism is unclear according to current knowledge.
[0127] Polypeptide Sequence:
TABLE-US-00008 (SEQ ID NO: 6) MPEKHDLLAAILAAKEQGIGAILAFAMAYLRGRYNGGAFTKTVIDATMC AIIAWFIRDLLDFAGLSSNLAYITSVFIGYIGTDSIGSLIKRFAAKKAG VEDGRNQ
[0128] S.sup.107: Wild-type of the S.sup.107 anti-pinholin of bacteriophage A. Analogously to the S.sup.21-71 anti-pinholin, the pore-forming properties are greatly limited due to two additional amino acids MK at the N-terminus. However, an exact mechanism is unclear according to current knowledge (Molecular Microbiology, 1993, 8, 525-33).
[0129] Polypeptide Sequence:
TABLE-US-00009 (SEQ ID NO: 7) MKMPEKHDLLAAILAAKEQGIGAILAFAMAYLRGRYNGGAFTKTVIDAT MCAIIAWFIRDLLDFAGLSSNLAYITSVFIGYIGTDSIGSLIKRFAAKK AGVEDGRNQ
[0130] S.sup.107-M3A: Mutant of the S.sup.107 anti-pinholin in which the second methionine is mutated to alanine at the third position. Thus, in principle, prevents the expression of the wild-type S.sup.105 holin, which in the case of S.sup.107 can also be expressed in principle by moving the translation initiation site by 3 amino acids. In comparison to the S.sup.107 anti-pinholin, the pore formation is thus further delayed.
[0131] Polypeptide Sequence:
TABLE-US-00010 (SEQ ID NO: 8) MKAPEKHDLLAAILAAKEQGIGAILAFAMAYLRGRYNGGAFTKTVIDAT MCAIIAWFIRDLLDFAGLSSNLAYITSVFIGYIGTDSIGSLIKRFAAKK AGVEDGRNQ
[0132] T4 pinholin: Wild-type of the T4 pinholin of bacteriophage T4 with highly pronounced pore-forming properties. The T4 pinholin has a transmembrane domain with a long periplasmic C-terminus and a short cytosolic N-terminus. According to the literature, both termini are regulated by an anti-holin mechanism, which runs completely differently than in the previously known holins S.sup.105 and S.sup.2168 (Journal of Bacteriology, 2016, 198, 2448-57). That is to say, no flipping of individual transmembrane helices is currently being postulated. However, currently, the exact mechanism remains unclear.
[0133] Polypeptide Sequence:
TABLE-US-00011 (SEQ ID NO: 9) MAAPRISFSPSDILFGVLDRLFKDNATGKVLASRVAVVILLFIMAIVWY RGDSFFEYYKQSKYETYSEIIEKERTARFESVALEQLQIVHISSEADFS AVYSFRPKNLNYFVDIIAYEGKLPSTISEKSLGGYPVDKTMDEYTVHLN GRHYYSNLKFAFLPTKKPTPEINYMYSCPYFNLDNIYAGTITMYWYRND HISNDRLESICAQAARILGRAK
[0134] T4.sup.ΔC-Tail: Truncated version of the T4 pinholin in which the periplasmically located C-terminus was deleted. This variant is non-toxic. Mechanism unknown so far.
[0135] Polypeptide Sequence:
TABLE-US-00012 (SEQ ID NO: 10) AAPRISFSPSDILFGVLDRLFKDNATGKVLASRVAVVILLFIMAIVWY
[0136] .sup.ΔN-TailT4.sup.ΔC-Tail: Truncated version of the T4 pinholin, which only has the transmembrane domain. This variant is toxic, mechanism unknown.
[0137] Polypeptide Sequence:
TABLE-US-00013 (SEQ ID NO: 11) MVAVVILLFIMAIVWY
Ion Channels
[0138] K.sub.CV.sup.NTS: Viral potassium channel that specifically conducts potassium, but also allows calcium to pass through. (BBA-Biomembranes, 2014, 1838, 1096-1103).
[0139] Polypeptide Sequence:
TABLE-US-00014 (SEQ ID NO: 12) MLLLIIHLSILVIFTAIYKMLPGGMFSNTDPTWVDCLYFSASTHTTVGY GDLTPKSPVAKLTATAHMLIVFAIVISSFTFPW
[0140] K.sub.CV.sup.NTS G77S: Based on K.sub.CV.sup.NTS with a mutation of glycine to serine at position 77. This mutation results in an average open probability and thus to a reduction in the conductivity of the potassium channel.
[0141] Polypeptide Sequence:
TABLE-US-00015 (SEQ ID NO: 13) MLLLIIHLSILVIFTAIYKMLPGGMFSNTDPTWVDCLYFSASTHTTVGY GDLTPKSPVAKLTATAHMLIVFAIVISGFTFPW
[0142] K.sub.CV.sup.PBCV1: Viral potassium channel that specifically conducts potassium, but also allows calcium to pass through. Very low open probability and thus low conductivity (FEBS Lett, 2003, 552, 12-6).
[0143] Polypeptide Sequence:
TABLE-US-00016 (SEQ ID NO: 14) MLVFSKFLTRTEPFMIHLFILAMFVMIYKFFPGGFENNFSVANPDKKAS WIDCIYFGVTTHSTVGFGDILPKTTGAKLCTIAHIVTVFFIVLTL
Others
[0144] HokB: Pore-forming membrane peptide; forms part of a bacterial efflux or toxin/antitoxin system. Specifically forms HokB nanopores in the inner membrane and thus enables the unspecific outflow of toxic substances, such as antibiotics (MBio. 2018, 9, pii: e00744-18).
[0145] Polypeptide Sequence:
TABLE-US-00017 (SEQ ID NO: 15) MKHNPLVVCLLIICITILTFTLLTRQTLYELRFRDGDKEVAALMACTSR
[0146] TisB: Pore-forming membrane peptide; forms part of a bacterial efflux or toxin/antitoxin system. Specifically forms TisB nanopores in the inner membrane and thus enables the unspecific outflow of toxic substances, such as antibiotics (Biophysical Journal, 2012, 103, 1460-1469).
[0147] Polypeptide Sequence:
TABLE-US-00018 (SEQ ID NO: 16) MNLVDIAILILKLIVAALQLLDAVLKYLK
[0148] αHLA: a-hemolysine refers to a pore-forming toxin from S. aureus (PNAS, 2008, 105, 19720-19725). Used as prototypic nanopore for a number of studies and projects in the area of nanopore engineering, especially in the development of sensory applications. Such projects are however almost exclusively realized by the expression of αHLA in cell-free systems. Functional states are investigated either by means of high-resolution biophysical methods or with blood cells as a substrate. Is in principle exported extracellularly, even in E. coli.
[0149] Polypeptide Sequence:
TABLE-US-00019 (SEQ ID NO: 17) MKIRIVSSVIIILLLGSILMNPVAGAADSDINIKIGIIDIGSNIIVKIG DLVIYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEG ANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFN GNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVI FNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSL LSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGT NTKDKWTDRSSERYKIDWEKEEMTN
[0150] cWZA: Minimum pore-forming membrane peptide artificially derived from the transmembrane region of the membrane protein Wza (Nature Chemistry, 2017, 9, 411-419). In reconstituted lipid membrane bilayers, cWZA has been shown to form defined heptameric pores; but relies on ring-shaped low-molecular substances that associate with the individual membrane peptides in order to form stable or defined nanopores in the membrane.
[0151] Polypeptide Sequence:
TABLE-US-00020 (SEQ ID NO: 18) MAPLVRWNRVISQLVPTITGVHDLTETVRYIKTWPN
[0152] BM2: Proton channel from the influenza B virus (Chemical Sciences, 2018, 9, 2365-2375).). Permeable exclusively to protons, depolarizes the inner membrane of E. coli, results in cell death. Used as control for checking the specificity of pore-related inflow of Ca.sup.2+ in various screening formats.
[0153] Polypeptide Sequence:
TABLE-US-00021 (SEQ ID NO: 19) MLEPFQILSISSFILSALHFIAWTIGHLNQIKR
[0154] HCV TME1 and TME2: Transmembrane domains of the hepatitis C virus shell proteins, which bind to the membrane and act toxically. (Biochim Biophys Acta, 2004, 28; 1660(1-2):53-65)
[0155] Polypeptide Sequences:
TABLE-US-00022 HCV TME1: (SEQ ID NQ: 20) MIAGAHWGVLAGIAYFSMVGNWAKVLVVLLLFAGVDA HCV TME2: (SEQ ID NO: 21) MEYVVLLFLLLADARVCSCLWMMLLISQAEA
Example 1: Validation of the Fluorescence Assay in Microtiter Plates
Background:
[0156] First, the newly developed method was validated in E. coli in microtiter plate format. In particular, there should be a clarification of the extent to which the inflow of Ca.sup.2+ as a function of the expression of a pore in the inner membrane of E. coli is associated with an increase in fluorescence. The model peptides were the pore-forming membrane peptide .sup.SGSΔTMD1-S.sup.2168 and the non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168 as positive or negative control.
Experimental Procedure and Results:
[0157] 1. Co-Transformation of the R-GECO1 and the Two Pinholin Fragments in Chemically Competent E. coli.
[0158] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pACYCT2-.sup.SGSΔTMD1-S.sup.2168 (positive)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pACYCT2-.sup.TVMVΔTMD1-S.sup.2168 (negative)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)
2. Inoculation Overnight:
[0159] Colonies of BL21(DE3) on LB agar from 1. were picked and cultivated in deep well plates with 300 μl of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 1300 rpm.
3. Microtiter Plate Readout:
[0160] After cultivating individual clones from 2. overnight, the OD.sub.600 of the precultures in microtiter plates (MTP) was adjusted to 0.1, and the samples, 200 μL each, were shaken at 37° C. and 180 rpm for 30 min.
[0161] Thereafter, the R-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, and the fluorescence was measured over 120 min.
[0162] Subsequently, the two pinholin fragments were then induced with 0.5 mM IPTG, and the fluorescence was measured over another 120 min.
[0163] General information: The measurement in the reader is carried out at 30° C. every 10 min. The plate is shaken at 180 rpm between the measurements. The OD.sub.600 nm and the fluorescence (Ex: 555 nm; Em: 600 nm) are measured. The volume in the cavities is 200 μl after addition of all inductors.
4. Experimental Analysis:
[0164] For the analysis, the data of three different colonies were averaged and the OD.sub.600 nm and in relative fluorescence units (RFU) were in each case plotted in a graph (
Conclusion:
[0165] Overall, pore-forming membrane peptides can be quantitatively imaged in microtiter plate format by means of the newly developed genetic assay in E. coli and can be differentiated from the non-pore-forming membrane peptides. Various parameters can be used to quantitatively characterize the pore-forming properties of a membrane peptide, a nanopore or an ion channel (
Example 2: Investigation of the Pore-Forming Properties of Various Membrane Peptides, Nanopores and Ion Channels in Microtiter Plates
Background:
[0166] It was then clarified whether or how easily the developed method can be applied to further pore-forming membrane peptides, nanopores and ion channels. For this purpose, we tested 19 different variants derived from a total of 10 different classes of nanopores or ion channels with respect to an expression-related increase in the fluorescent signal in E. coli. Measurements were basically carried out in the microtiter plate format. The results are summarized (
Experimental Procedure and Results:
[0167] 1. Co-Transformation of the R-GECO1 and the Pore-Forming Proteins in Chemically Competent E. coli.
[0168] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pTeT7W-“nanopore” (Table 1).
2. Inoculation Overnight:
[0169] Colonies of BL21(DE3) on LB agar from 1. were picked and cultivated in deep well plates with 300 μl of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 1300 rpm.
3. Microtiter Plate Readout:
[0170] After cultivating individual clones from 2. overnight, the OD.sub.600 of the precultures in microtiter plates (MTP) was adjusted to 0.1, and the samples, 200 μL each, were shaken at 37° C. and 180 rpm for 30 min.
[0171] Thereafter, the R-GECO1 was initially induced with 25 mM sodium 25 propionate, pH 8.0, and the fluorescence was measured over 120 min.
[0172] Subsequently, the pores were then induced with 0.5 mM IPTG, and the fluorescence was measured over another 120 min.
[0173] General information: The measurement in the reader is carried out at 30° C. every 10 min. The plate is shaken at 180 rpm between the measurements. The OD.sub.600 nm and the fluorescence (Ex: 555 nm; Em: 600 nm) are measured. The volume in the cavities is 200 μl after addition of all inductors.
4. Experimental Analysis:
[0174] For the analysis, the data of three different colonies were averaged and the OD.sub.600 nm and in the fluorescence in relative fluorescence units (RFU) were in each case plotted in a graph (
Conclusion:
[0175] In summary, a total of 20 variants of 10 different nanopores, ion channels and membrane peptides were successfully measured with the method (
TABLE-US-00023 TABLE 1 Overview of the various classes of nanopores and ion channels that were investigated by means of the novel method. Nanopore Nanopore System Variant Results/Comments S.sup.21 S.sup.21-68 Wild-type S21-68 pinholin. Shows pronounced pore formation. S.sup.21-71 Shows limited pore formation in comparison to S.sup.21-68. S.sup.21-71 M4A M4A mutation prevents expression of the wild-type S.sup.21-68 pinholin moving the translation initiation screen and thus shows greatly limited pore formation in comparison to S.sup.21-68 and S.sup.21-71. SGS- The second transmembrane .sup.ΔTMD1S.sup.21 domain of the S.sup.21-68 pinholin shows pronounced pore formation. TVMV- A preceding TVMV interface .sup.ΔTMD1S.sup.21 severely limits pore formation. S.sup.105 S.sup.105 Wild-type holin of bacteriophage A with pronounced pore formation. S.sup.107 Wild-type anti-holin of bacteriophage A with limited pore-forming properties. S.sup.107-M3A M3A mutation prevents expression of the wild-type S105 pinholin moving the translation initiation screen and thus shows greatly limited pore formation in comparison to S105 and S107. T4 holin Wild-type Wild-type holin of the bacteriophage T4 with pronounced pore formation. T4.sup.ΔC-Tail Truncated version of the T4 holin in which the periplasmically positioned C-terminus was deleted, with limited pore formation. .sup.ΔN-TailT4.sup.ΔC-Tail Truncated version of the T4 holin, which only has the transmembrane domain, with strongly pronounced pore formation. K.sub.CV PBCV1 Variant of a K.sup.+-specific ion channel with a promiscuous permeability to Ca.sup.2+ depending on the opening probability of the ion channel NTS G77S Variant of a K.sup.+-specific ion channel with a promiscuous permeability to Ca.sup.2+ depending on the opening probability of the ion channel NTS Variant of a K.sup.+-specific ion channel with a promiscuous permeability to Ca.sup.2+ depending on the opening probability of the ion channel Nanopores HokB Pore-forming membrane peptide shows from toxin/ pronounced pore-forming properties. antitoxin TisB Pore-forming membrane peptide shows systems pronounced pore-forming properties. αHLA Pore-forming toxin, but does not result in pore formation within the scope of the method. Presumably because is it directly exported from E. coli via defined export signals: i.e., there is no canonical export sequence (e.g., no PelB leader sequence). cWZA Artificial pore-forming membrane peptide, which forms artificial nanopores only in the context of in-vitro lipid bilayer experiments, but does not result in functional pore formation in the context of the genetic screening method. BM2 NEGATIVE CONTROL: Pore-forming membrane peptides derived from the influenza B virus and only permeable to protons. Demonstrably results in cell death on account of the pore formation, but as expected not in an increase in the Ca2+-dependent fluorescence. HCV TME1 Transmembrane domains of the hepatitis TMEs C virus shell proteins, which bind to the membrane and act toxic TME2 Transmembrane domains of the hepatitis C virus shell proteins, which bind to the membrane and act toxic
Example 3: Model Selection Experiments in Microtiter Plates
Background:
[0176] An important aspect in the development of screening methods is to perform a check of the extent to which a functional variant of a larger amount of non-functional variants can be identified, selected and enriched. This is usually detected in model selections in which the DNA of a functional protein is diluted relative to a non-functional protein and then reidentified by means of a functional assay.
[0177] In the course of a model selection, it is thus comprehensively demonstrated that the connection between genotype and phenotype, i.e., the physical association or assignment between the function of a protein and its coding DNA is maintained during the entire screening cycle. In the context of pore-forming membrane peptides, there must also be a check as to what extent toxic effects associated with the pore-forming properties of the membrane peptides, for example during growth due to non-specific expression, i.e., before they are subjected to a functional test, are negatively enriched or a selection against the desired properties takes place.
[0178] The model peptides were, as already in Example 1, .sup.SGSATMD1-S.sup.2168 as pore-forming and .sup.TVMVATMD1-S.sup.2168 as non-pore-forming membrane peptide.
Experimental Procedure and Results:
[0179] 1. Co-Transformation of the R-GECO1 and the Two Pinholin Fragments in Chemically Competent E. coli. The R-GECO1 could be Expressed as Previously in pPRO24 and the Pinholin Fragments in pTeT7W.
[0180] The following combinations were tested as dilution in each case:
.fwdarw.In a ratio of 1:5 of .sup.SGSΔTMD1-S.sup.2168 to .sup.TVMVΔTMD1-S.sup.2168+R-GECO1
.fwdarw.In a ratio 1:10 of .sup.SGSATMD1-S.sup.2168 to .sup.TVMVΔTMD1-S.sup.2168+R-GECO1
2. Inoculation Overnight:
[0181] Colonies with ratios of 1:5 and 1:10 were randomly picked and cultivated in 25 deep well plates with 500 μl of LB medium with the necessary antibiotics overnight for about 16 h at 30° C. and 1300 rpm.
[0182] For this purpose, 89 colonies were picked in each case
[0183] In addition, per 96-well microtiter plate, 3 positive controls and 3 negative controls were picked in a targeted manner
[0184] 1 cavity was filled only with LB medium as control
[0185] The deep well plate was stored at 4° C.
3. Preparation for Microtiter Plate Readout:
[0186] After cultivation from 2. overnight, the OD.sub.600 of the precultures in microtiter plates (MTP) was adjusted to about 0.1, and the samples, 200 μL each, were shaken at 37° C. and 180 rpm for 30 min. It was assumed that all others have a similar OD.sub.600 nm and that all samples can thus be diluted identically.
[0187] Thereafter, the R-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, and the fluorescence was measured over 120 min.
[0188] General information: The measurement in the reader is carried out at 30° C. every 10 min. The plate is shaken at 180 rpm between the measurements. The OD.sub.600 nm and the fluorescence (Ex: 555 nm; Em: 600 nm) are measured. The volume in the cavities is 200 μl.
4. Experimental Analysis
[0189] For the analysis, the data of each cavity, i.e., the OD.sub.600 nm and the fluorescence in relative fluorescence units (RFU) were plotted separately in each case. For normalization and better clarity, the fluorescence can also be divided by the OD.sub.600 independently of the toxicity. For a more clearly arranged analysis, end point measurements are shown below:
.fwdarw.120 min after sodium propionate, but before IPTG addition and induction of the pore (
.fwdarw.120 min after IPTG addition and induction of the pore (
[0190] Subsequently, the clones with the highest fluorescence values were reinoculated in the deep well plate, which was stored at 4° C., and the DNA was isolated. In all colonies, sequencing resulted in the positive control pACYCT2-.sup.SGSΔTMD1-S.sup.2168.
.fwdarw.In the 1:5 dilution (
.fwdarw.In the 1:10 dilution (
[0191] Optionally, it is possible to change the threshold value in both model selections and thus to select for different properties. In addition, the kinetics (i.e., the half-maximum time of the fluorescence signal or the slope) can also be analyzed during the pore formation, which is visible immediately after induction of the nanopore.
Conclusion:
[0192] Overall, by means of the newly developed method, the DNA of the pore-forming membrane peptide .sup.SGSΔTMD1-S.sup.2168 can be reidentified from a five- or ten-fold dilution to the non-pore-forming peptide .sup.TVMVΔTMD1-S.sup.2168 in correspondingly equal proportions within a selection cycle in the microtiter plate format, selected and thus enriched. On the one hand, this demonstrates that the connection between genotype and phenotype is maintained within the selection cycle and that under the given circumstances, no counter-selection of the desired properties takes place, for example due to the toxicity of the pore-forming peptides.
Example 4: Selection Experiments for Finding New Properties and Functions of Pore-Forming Membrane Peptides in Microtiter Plates
Background:
[0193] In addition to model selections, a library of pore-forming variants of the wild-type pinholin S.sup.2168 in the microtiter plate format was also screened for new properties or functions. The primary aim of a real selection is to perform a check of the extent to which a screening method can be used to identify 30 novel properties or functions of pore-forming membrane peptides. In addition, i.e., analogously to the model selection experiments with the two pinholin variants .sup.TVMVΔTMD1-S.sup.2168 and .sup.SGSATMD1-S.sup.2168 (see Example 3), there must be a check of the extent to which the pore-forming properties in the context of a larger library have an effect on the relative frequency of variants during growth, i.e., before a functional test is carried out.
[0194] Specifically, the three N-terminal amino acids of the natural S.sup.2171 pinholin were randomized by means of saturation mutagenesis in order to check which mutations have a positive or negative effect on the pore-forming properties of the wild-type S.sup.2171 pinholin.
Experimental Procedure and Results:
1. Design of the Mutation Primer
[0195] The mutagenesis primer for the S.sup.2171 anti-holin designed for the following base pair sequence or amino acid sequence:
TABLE-US-00024 Amino acids: ATG AAA TCT ATG GAC AAA ATC TCA ACT M K S M D K I S T Primer: (SEQ ID NO: 22) TGATGAGG CAT ATG NNK NNK NNK GAC AAA ATC TCA ACT
2. Cloning of the Library
[0196] Initially, the S.sup.2171 anti-holin was amplified by the above-mentioned forward primer and a suitable reverse primer and brought into the pCTRL2 vector with the respective interfaces NdeI/KpnI. A small model library was prepared from about 4000 clones (DH10β).
3. Co-transformation of the library in chemically competent BL21(DE3)
[0197] Transformed individually in each case
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pCTRL2-.sup.SGSΔTMD1-S.sup.2168 (positive)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pCTRL2-.sup.TVMVΔTMD1-S.sup.2168 (negative)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (Reporter)+pCTRL2-S.sup.2168
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pCTRL2-S.sup.2171
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pCTRL2-S.sup.2171 M4A
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pCTRL2-.sup.3×NNKS.sup.2168 (library)
[0198] Only as a result of the specially developed pCTRL2 vector could a retransformation of the wild-type holin to this extent be ensured.
4. Inoculation Overnight:
[0199] Colonies of BL21(DE3) on LB agar from 3. were picked and cultivated in deep well plates with 300 μl of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 1300 rpm.
5. Microtiter Plate Readout:
[0200] After cultivating individual clones from 2. overnight, the OD.sub.600 of the precultures in microtiter plates (MTP) was adjusted to 0.1, and the samples, 200 μL each, were shaken at 37° C. and 180 rpm for 30 min.
[0201] Thereafter, the R-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, and the fluorescence was measured over 120 min.
[0202] Subsequently, the library along with the positive and negative control were then induced with 0.5 mM IPTG, and the fluorescence was measured over another 120 min.
[0203] General information: The measurement in the reader is carried out at 30° C. every 10 min. The plate is shaken at 180 rpm between the measurements. The OD.sub.600 nm and the fluorescence (Ex: 555 nm; Em: 600 nm) are measured. The volume in the cavities is 200 μl after addition of all inductors.
6. Analysis
[0204] For the analysis, the curves were fitted empirically with equation 1 in order to quantitatively determine the half-maximum time c or the slope d, for example. The distribution of the parameters investigated with respect to the half-maximum time c and the slope d of the library is summarized by way of example in
[0205] Individual mutants are subsequently sequenced and subjected to a detailed functional characterization. In this case, five individually investigated mutants show different pore-forming properties depending on the N-terminal sequences (
Conclusion:
[0206] In addition, the screening of a part of the library shows that changes in the N-terminal sequence before the first transmembrane domain (TMD) can change the pore-forming properties of the S.sup.21-68 pinholin. First structure/function correlations indicate that charges have a strong effect on the slope and the half-maximum time of the pore formation. The high transformation efficiency of the libraries of >10.sup.5 per μg DNA indicates that sufficiently large libraries of a pore-forming membrane peptide that is in principle toxic can routinely be transformed in E. coli and its pore-forming properties can be read by means of the optical reporters R-GECO1 and G-GECO1. If necessary, the expression of the pore-forming peptides in the non-induced state can be further suppressed.
Example 5: Obtaining the Cellular Integrity or Extent of the Cell Lysis after Expression of Pore-Forming Membrane Peptides
Background:
[0207] In principle, it is not possible to perform a check in the microtiter plate as to what extent the cell lysis takes place in each case after expression of a pore-forming membrane peptide or to what extent the cellular integrity of the 10 E. coli is preserved. Both aspects have advantages and disadvantages or develop different biotechnological applications: (a) In order to screen pore-forming membrane peptides at the single-cell level, for example by means of a flow cytometer or in the context of a microfluidic system, it is critical that the cellular integrity is preserved in order to maintain the connection between genotype and phenotype during the entire selection cycle; (b) Alternatively, the loss of cellular integrity and associated cell lysis can be used in biotechnological processes to release the cytosolic content of a microorganism, e.g., in the recombinant expression of proteins, or also in industrial biotechnology to release important metabolic products on an industrial scale.
[0208] Specifically, there was an investigation as to what extent the expression of a pore-forming peptide lyzes cells or to what extent the cellular integrity in the form of a shell is preserved. This was demonstrated by the proportion of fluorescent proteins in the soluble fraction in comparison to the non-soluble fraction. Soluble protein is conditional on digestion of the cell, wherein fluorescence in the insoluble fraction is due to cells in which cellular integrity is preserved.
Experimental Procedure and Results:
[0209] 1. Co-Transformation of R-GECO1 and the Lyzing and Non-Lyzing Pores in Chemically Competent E. coli.
[0210] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pTeT7W-.sup.SGSΔTMD1-S.sup.2168 (non-lyzing)
.fwdarw.BL21(DE3) with PRO24-R-GECO1 (reporter)+pTeT7W-BM2 (non-lyzing)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pTeT7W-S.sup.2171 (semi-lyzing)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pTeT7W-S.sup.107 (lyzing)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pTeT7W-T4 (lyzing)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pTeT7W-TisB (lyzing)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter)+pTeT7W-HokB (lyzing)
.fwdarw.BL21(DE3) with pPRO24-R-GECO1 (reporter) (negative control)
2. Inoculation Overnight:
[0211] Colonies of BL21(DE3) on LB agar from 1. were picked and cultivated in deep well plates with 300 μl of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 1300 rpm.
3. Sample Preparation: Semi-Denaturing SDS-PAGE Gel
[0212] After cultivating individual clones from 2. overnight, the OD.sub.600 of the precultures in 24-cavity plates was adjusted to 0.1, and the samples, 2 ml each, were shaken at 37° C. and 180 rpm for 30 min.
[0213] Thereafter, the R-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, at 30° C. and 180 rpm for 120 min.
[0214] Subsequently, the pores were then induced with 0.5 mM IPTG at 30° C. and 180 rpm for 180 min.
4. Analysis: Semi-Denaturing SDS-PAGE Gel
[0215] After 180 min, 20 μl of each sample was collected and the supernatant was separated from the pellet by centrifugation
[0216] The pellet was dissolved in 20 μl resuspension buffer (200 mM Na.sub.3PO.sub.4, 1 mM CaCh) and a further 20 μl 2×SDS buffer was added (total volume 40 μl)
[0217] 20 μl 2×SDS buffer (+1 mM CaCh) was added to the supernatant (total volume 40 μl)
[0218] General information: 12% SDS-PAGE gel, run at 120 V for about 110 min. Gels were recorded using the Amersham Imager600 gel documentation unit (extinction: 520 nm; emission 605/BP40 nm). All gels were exposed identically to guarantee comparability.
Conclusion:
[0219] Overall, different pores under the given expression conditions show different propensities to preserve cellular integrity or to lyze the cells. Under exponential growth after 3 h (
Example 6: Assay Validation for the Screening of Pore-Forming Membrane Peptides in Individual Colonies on Agar Plates
Background:
[0220] Alternatively, the newly developed assay can also be applied in colony format on agar plates. In comparison to microtiter plates (see Examples 1-5), screening methods based on colonies enable higher throughput and, moreover, do not rely on expensive laboratory equipment, such as a microtiter plate spectrophotometer. Specifically, there must be a check of the extent to which quantitative analysis or differentiation of pore-forming peptides from non-pore-forming peptides is also possible at the level of individual colonies. The model peptides were, as before, the pore-forming membrane peptide .sup.SGSΔTMD1-S.sup.2168 and the non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168 as positive or negative control.
Experimental Procedure and Results:
[0221] 1. Co-Transformation of the R-GECO1 and the Two Pinholin Fragments in Chemically Competent E. coli.
[0222] The following combinations were tested:
.fwdarw.BL21(DE3) with pACYCT2-ΔTMD1-S.sup.2168+pPRO24-R-GECO1
.fwdarw.BL21(DE3) with pACYCT2-.sup.TVMVΔTMD1-S.sup.2168+pPRO24-R-GECO1
[0223] The transformation batches are coated on LB agar with appropriate 25 antibiotics and cultivated overnight at 37° C.
2. Procedure the Colony-Based Assay:
[0224] On the following day, the colonies are transferred with a filter paper to a 30 further LB agar plate with 25 mM sodium propionate in order to express R-GECO1.
[0225] The stamped plate is cultivated for 4 h at 37° C. The expression of the pinholin fragments is subsequently induced with 0.5 mM IPTG
[0226] For this purpose, a filter paper is sprayed with the corresponding solution and placed on the colonies for about 10 s
[0227] The plate is cultivated for another 2 h at 37° C. until subsequent storage at 4° C. until the following day (with light protection)
[0228] On the following day, the fluorescence of the colonies is recorded at the extinction wavelength of 520 nm (0.3 s exposure time)
3. Experimental Analysis:
[0229] The fluorescence of at least >180 colonies in each case is shown in
Conclusion:
[0230] The newly developed method can also be used in the colony format on agar plates to differentiate pore-forming membrane peptides from non-pore-forming membrane peptides. Significant differences in the fluorescent signal between individual colonies can be observed after a single time as a function of the expression of pore-forming .sup.SGSΔTMD1-S.sup.21 68 or non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168 (
Example 7: Model Selection for the Screening of Pore-Forming Membrane Peptides in Colonies on Agar Plates
Background:
[0231] Analogously to the procedure on microtiter plates, there was a check in model selections as to what extent the connection between genotype and phenotype is preserved even in individual colonies on agar plates within the entire selection cycle, and the DNA of a pore-forming membrane peptide can be reidentified from a larger quantity of non-pore-forming membrane peptides, selected and enriched. Analogously to the validation of the method in microtiter plates, screening for colonies enables higher throughput and does not rely on expensive microtiter plate spectrophotometers. The model peptides were, as before, the pore-forming membrane peptide .sup.SGSΔTMD1-S.sup.2168 and the non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168, again as positive or negative control.
Experimental Procedure and Results:
[0232] 1. Co-Transformation of the R-GECO1 and the Two Pinholin Fragments in Chemically Competent E. coli.
[0233] The following combinations were tested as dilution in each case:
.fwdarw.In the ratio 1:5 of ATMD1-S.sup.2168 to .sup.TVMVΔTMD1-S.sup.2168+R-GECO1
.fwdarw.In the ratio 1:10 of ATMD1-S.sup.2168 to .sup.TVMVΔTMD1-S.sup.2168+R-GECO1
[0234] The transformation batches are coated on LB agar with appropriate antibiotics and cultivated overnight at 37° C.
2. Procedure for the Colony-Based Assay:
[0235] On the following day, the colonies are transferred with a filter paper to a further LB agar plate with 25 mM sodium propionate in order to express R-GECO1
[0236] The stamped plate is cultivated for 4 h at 37° C. The expression of the pinholin fragments is subsequently induced with 0.5 mM IPTG. For this purpose, a filter 30 paper is sprayed with the corresponding solution and placed on the colonies for about 10 s.
[0237] The plate is cultivated with light protection for another 2 h at 37° C. until subsequent storage at 4° C. until the following day.
[0238] On the following day, the fluorescence of the colonies is recorded at the extinction wavelength of 460 nm (0.1 s), 520 nm (1 s) and 630 nm (1 s)
3. Analysis:
[0239] For the determination of the colonies with the highest fluorescence, only the data from the channel at 520 nm are used.
[0240]
Conclusion:
[0241] In summary, the pore-forming membrane peptides can be (a) differentiated quantitatively on agar plates from the non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168 and (b) selected and enriched from a greater dilution of 1:5 and 1:10 (
Example 8: Assay Validation at the Single-Cell Level by Means of a Flow Cytometer
Background:
[0242] Alternatively, the newly developed assay can also be used in order to investigate the pore-forming properties or functions of membrane peptides at the single-cell level by means of a flow cytometer. In comparison to microtiter 30 plates (see Examples 1-5) or also colonies on agar plates (see Examples 6 and 7), this has the advantage that the assay can be measured with high resolution and with a high statistical significance: >10.sup.6 variants within one FACS measurement. Specifically, there must be a check of the extent to which quantitative analysis or differentiation of pore-forming peptides from non-pore-forming peptides is also possible at the level of individual cells. The model peptides were, as before, the pore-forming membrane peptide .sup.SGSΔTMD1-S.sup.2168 and the non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168, again as positive or negative control.
Experimental Procedure and Results:
1. Co-Transformation of the Library in Chemically Competent BL21(DE3)
[0243] Transformed individually in each case
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pTeT7W-.sup.SGSΔTMD1-S.sup.2168 (positive)
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pTeT7W-.sup.TVMVΔTMD1-S.sup.2168 (negative)
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pTeT7W-.sup.SGSΔTMD1-S.sup.2168 (positive)+pTeT7W-.sup.TVMVΔTMD1-S.sup.2168 (negative) in the ratio 1:10 of positive to negative
2. Inoculation Overnight:
[0244] Colonies of BL21(DE3) on LB agar from 1. were picked and cultivated in 25 culture tubes with 2 ml of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 220 rpm.
[0245] Mixed variants of 1:10 and 1:100 from positive to negative were not plated, but were transferred directly after transformation into medium and allowed to grow
3. FACS Sample Preparation:
[0246] After cultivation from 2. overnight, the OD.sub.600 of the precultures in culture tubes was adjusted to about 0.1, and the samples, 2 ml each, were shaken at 37° C. and 180 rpm for 30 min.
[0247] Thereafter, the G-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, cultivated at 30° C. and 220 rpm for 120 min.
[0248] Subsequently, the positive and negative control, as well as the 1:10 and 1:100 mixed cultures were then induced with 0.5 mM IPTG at 30° C. and 220 rpm for 60 min.
[0249] After renewed measurement of the OD.sub.600 nm, the cultures were washed with 2×PBS, subsequently adjusted with PBS to an OD.sub.600 nm of 0.05 and stored on ice
4. FACS Measurements
[0250] The fluorescence measurements on the FACS (SONY SH800S) were carried out with an extinction of 480 nm and an emission of 525 nm. The assay validation results are summarized in
Conclusion:
[0251] In summary, in flow-cytometric analysis, two differently fluorescing populations of E. coli result (depending on the expression of a pore-forming .sup.SGSATMD1-S.sup.2168 in comparison to a non-pore-forming peptide .sup.TVMVATMD1-S.sup.2168). In parallel, a separation into forward and backward scatters indicates that the cellular integrity after expression of the pore-forming peptide is largely preserved and that it is thus possible, i.e., the method is in principle suited, to differentiate membrane peptides having different pore-forming properties at the single-cell level by flow cytometry.
Example 9: Model Selection at the Single-Cell Level by Means of a Flow Cytometer
Background:
[0252] Alternatively, the newly developed assay can be used to sort and enrich the pore-forming properties or functions of membrane peptides at the level of individual cells by means of a flow cytometer. In comparison to microtiter plates (see Examples 1-5) or also colonies on agar plates (see Examples 6 and 7), this has the advantage that the assay can be carried out quantitatively with a very high throughput, which in principle makes it possible to screen >10.sup.6 variants within one selection cycle. Specifically, there must be a check of the extent to which quantitative analysis or differentiation of pore-forming from non-pore-forming peptides is also possible at the level of individual cells. For this purpose, the already known transmembrane peptides .sup.SGSΔTMD1-S.sup.2168 and .sup.TVMVΔTMD1-S.sup.2168 served as positive or negative control.
Experimental Procedure and Results:
1. Co-Transformation of the Peptides in Chemically Competent BL21(DE3)
[0253] Transformed individually in each case
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2-.sup.SGSΔTMD1-S.sup.2168 (positive)
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2-.sup.TVMVΔTMD1-S.sup.2168 (negative)
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2-.sup.SGSΔTMD1-S.sup.2168 (positive)+pCTRL2-.sup.TVMVΔTMD1-S.sup.2168 (negative) in the ratio 1:10 of positive to negative
2. Inoculation Overnight:
[0254] Colonies of BL21(DE3) on LB agar from 1. were picked and cultivated in culture tubes with 2 ml of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 220 rpm.
[0255] Mixed variants of 1:10 and 1:100 from positive to negative were not plated, but were transferred directly after transformation into medium and allowed to grow
3. FACS Sample Preparation:
[0256] After cultivation from 2. overnight, the OD.sub.600 of the precultures in culture tubes was adjusted to about 0.1, and the samples, 2 ml each, were shaken at 37° C. and 180 rpm for 30 min.
[0257] Thereafter, the G-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, cultivated at 30° C. and 220 rpm for 120 min.
[0258] Subsequently, the positive and negative control, as well as the 1:10 mixed cultures were then induced with 0.5 mM IPTG at 30° C. and 220 rpm for 60 min.
[0259] After renewed measurement of the OD.sub.600 nm, the cultures were washed with 2×PBS, subsequently adjusted with PBS to an OD.sub.600 nm of 0.05 and stored on ice
4. FACS Measurements
[0260] The fluorescence measurements on the FACS (SONY SH800S) were carried out with an extinction of 480 nm and an emission of 525 nm. The results of the model selection experiments on the FACS are summarized in
Conclusion:
[0261] In summary, the pore-forming membrane peptide .sup.SGSΔTMD1-S.sup.2168 can 30 be (a) differentiated quantitatively at the single-cell level from the non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168 and (b) selected and enriched from a greater dilution of 1:10. It should be noted that depending on the expression of a pore-forming peptide, in this case .sup.SGSΔTMD1-S.sup.2168, the cell integrity of the cell is preserved to the extent that selection by FACS is possible.
Example 10: Validation of a Method for Screening Molecular Sensors and Switches
Background:
[0262] In addition to the development of a method for screening pore-forming membrane peptides, nanopores and ion channels (see Examples 1-9), the method can be reconfigured in order to optimize molecular sensors and switches in combination. Specifically, the challenge of developing methods for screening protein sensors or switches is to measure the molecular state of a sensor in each case with and without a ligand (and thus to select for the highest possible differential in the signal). It is critical and technically demanding to supply a defined amount of a ligand to the sensor or switch in order to activate the latter.
[0263] For this purpose, the sensor or switch can be exported extracellularly or periplasmically, for example. In these cases, an externally added ligand can associate with the sensor and switch comparatively easily. This also applies in the case of periplasmically exported sensors and switches since the outer E. coli membrane is comparatively permeable to a large number of low-molecular substances. However, this is only possible to a limited extent for fluorescent protein sensors consisting of a plurality of independently folding domains and also limits the folding properties and stability of the sensor or switch. Alternatively, in the case of cytosolic expression, the challenge is to allow the ligands to flow into or out of the cell via the membrane. Due to the intrinsically impermeable membrane, this strongly limits the number and nature of the ligands, however.
[0264] As part of the underlying method, the expression of nanopores is now to enable an increased permeability of the inner E. coli membrane via the expression of nanopores. The key development step is to investigate the extent to which low-molecular substances that are many times larger than Ca.sup.2+ ions can diffuse through nanopores of different sizes. Specifically, the R- and G-GECOs are to be replaced by L-Glu-specific fluorescence sensors, so-called iGluSnFRs, in order to detect the inflow and outflow of L-Glu beyond Ca.sup.2+ ions.
Experimental Procedure and Results:
[0265] 1. Co-Transformation of iGluSNFr and the Two Pinholin Fragments in Chemically Competent E. coli.
[0266] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-.sup.SGSΔTMD1-S.sup.2168 (positive)
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-.sup.TVMVΔTMD1-S.sup.2168 (negative)
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-BM2 (negative)
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-S.sup.2171-M4A
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-S.sup.2171-M3A
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-HokB
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-K.sub.CV.sup.NTS
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-K.sub.CV.sup.G77S
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)+pCTRL2-K.sub.PBCV1
.fwdarw.BL21(DE3) with pPRO24-iGluSNFr (reporter)
2. Inoculation Overnight:
[0267] Colonies of BL21(DE3) on LB agar from 1. were picked and cultivated in deep well plates with 300 μl of M9 medium (defined medium without glutamate) with the necessary antibiotics overnight for about 16 h at 37° C. and 1300 rpm.
3. Microtiter Plate Readout:
[0268] After cultivating individual clones from 2. overnight, the OD.sub.600 of the precultures in microtiter plates (MTP) was adjusted to 0.1, and the samples, 200 μL M9 medium each, were shaken at 37° C. and 180 rpm for 30 min.
[0269] Thereafter, the iGluSNFr was initially induced with 25 mM sodium propionate, pH 8.0, and the fluorescence was measured over 290 min.
[0270] Subsequently, the two pore-forming peptides were then induced with 0.5 mM IPTG, and the fluorescence was measured over another 260 min.
[0271] General information: The measurement in the reader is carried out at 30° C. every 10 min. The plate is shaken at 180 rpm between the measurements. The OD.sub.600 nm and the fluorescence (Ex: 480 nm; Em: 525 nm) are measured. The volume in the cavities is 200 μl after addition of all inductors.
Conclusion:
[0272] Overall, the expression of the pore-forming membrane peptide .sup.SGSΔTMD1-S.sup.2168 results in a reduction of the fluorescent signal of the iGluSNFr; in contrast, after expression of the non-pore-forming membrane peptide .sup.TVMVΔTMD1-S.sup.2168, the iGluSNFr-related signal remains unchangedly high (results combined in each case in
Example 11—Validation of the Method in a Microfluidic Experiment Arrangement
Background:
[0273] Microfluidic methods enable high-resolution functional studies, in particular in combination with optical measuring methods, for example based on fluorescence. Microfluidic methods offer a number of technical advantages, such as miniaturization of bioanalytical methods (Journal of Laboratory Automation, 2013, 18, 350-66), temporally resolved dynamic studies of microorganisms at the single-cell level in a microfluidic cultivation chamber (see examples in the respective review articles, Journal of Molecular Biology, 431, 2019, 4569-4588) or possibilities for screening in high throughput in droplet-based compartments with individual cells or in microcolonies (see examples in the respective review articles) Current Opinion Structural Biology, 2018, 48, 149-156, and Current Opinion in Chemical Biology, 2017, 37, 137-144 and specifically ACS Synthetic Biology, 2017, 6, 1988-1995).
[0274] In this context, the pore-forming properties of two different membrane proteins (specifically of the S.sup.2168 pinholin and T4 holin) were investigated in a microfluidic cultivation chamber on a single-cell level in temporally resolved form with a fluorescence microscope. In addition and as a continuation of Example 5, there was also an investigation as to what extent the expression of a pore-forming membrane protein lyzes the cells or to what extent the cellular integrity in the form of a shell is preserved.
Experimental Procedure and Results:
[0275] 1. Co-Transformation of G-GECO1 and the Nanopore Variants in Chemically Competent E. coli.
[0276] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.sRBS-S.sup.2168
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.T4
2. Inoculation Overnight:
[0277] Colonies of BL21(DE3) on LB agar plates from 1. were picked and cultivated in culture tubes with 2 ml of LB medium and the necessary antibiotics overnight for about 16 h at 37° C. and 180 rpm.
3. Microfluidics Readout:
[0278] After the cultivation of individual clones from 2. overnight, the cells were transferred to fresh sterile-filtered LB medium, the OD.sub.600 of the precultures was 20 adjusted to 0.1, and subsequently grown at 37° C. for a period of 3 h up to an OD.sub.600 of 2.
[0279] For the cultivation and functional fluorescence-microscopic examination of the cells in a microfluidic cultivation chamber, it was possible to use a previously published microfluidic chip design (see Supplementary
[0280] After the microfluidic chip was flooded with LB medium, the cells were added and the flow rate through the microfluidic chip was adjusted to 1 μl/min. The cells were left in the microfluidic chip at 37° C. for 3 h, so that they were able to collect and grow accordingly in the cultivation chambers.
[0281] Thereafter, the expression of G-GECO1 was induced by a change to LB medium with 25 mM sodium propionate, pH 8.0, at 30° C. for 2 h.
[0282] Subsequently, the nanopores were then induced by a change to LB medium with 0.5 mM IPTG, and the development of the fluorescent signal was measured over 16 h.
[0283] The fluorescent signal was measured by means of a fluorescence microscope at 30° C. in intervals of 3 minutes. A transmitted-light image and then a fluorescence image (Ex: 488 nm; Em: 504-539 nm) in the form of a Z-stack were taken each time. From the induction of G-GECO1, a flow rate of 4 μl/min was used.
[0284] Image sections of the fluorescence-microscopic images in the microfluidic cultivation chamber are shown by way of example for the S.sup.2168 pinholin at four different points in time (
4. Experimental Analysis:
[0285] For the analysis, the entire cells from a single cultivation chamber (shown by way of example in
Conclusions:
[0286] In summary, the test series demonstrates the application of the method in a microfluidic device. Specifically, cells were cultivated in a microfluidic cultivation chamber, and the pore-forming properties of the S.sup.2168 pinholin and the T4 holin were investigated at the single-cell level in temporally resolved form under a fluorescence microscopy.
[0287] As a function of different membrane proteins, different fluorescent signals resulted: The expression of the S.sup.2168 pinholin causes the cell division to stop quickly and the fluorescent signal to increase quickly (
[0288] In addition to Example 5, the results achieved show that the “cellular container” remains intact despite pore-forming properties, and inflow of Ca.sup.2+ ions into the cell occurs as a function of the nanopore. For comparatively large nanopores, such as the T4 holin, the time window is comparatively short (in this case 30 min) before either a loss of cellular integrity or outflow of the sensor occurs (
Example 12—Selection Experiments for Finding New Properties and Functions of Pore-Forming Membrane Peptides in Microtiter Plates
Background:
[0289] In addition to model selections, a library of pore-forming variants of the S.sup.2171 pinholin in the microtiter plate format was also screened for changed pore-forming properties. The primary goal of a real selection is to perform a check of the extent to which the method can be used to select membrane proteins with novel pore-forming properties and functions.
[0290] Specifically, the three N-terminal amino acids of the S.sup.2171 pinholin were randomized by means of saturation mutagenesis in order to check which mutations have a positive or negative effect on the pore-forming properties of the wild-type S.sup.2171 pinholin. In addition, there should be a clarification of the extent to which the promoter strength, measured at the half-maximum time c, and the slope d have an influence on the dynamic range of the method in the context of a library selection.
Experimental Procedure and Results:
1. Design of the Mutagenesis Primer:
[0291] The mutagenesis primer for the S.sup.2171 pinholin designed for the following DNA sequence or amino acid sequence:
[0292] The randomized codons in the context of the N-terminally randomized pinholin library .sup.3×NNKS.sup.2168 are in each case denoted by X.sub.1, X.sub.2 and X.sub.3 or position 1, position 2 and position 3.
2. Cloning of the Library
[0293] Initially, the S.sup.2171 pinholin was amplified by the above-mentioned forward primer and a suitable reverse primer and cloned with the respective interfaces for NdeI/KpnI into the pCTRL2 or pCTRL2.T7.100.sRBS vector.
[0294] A library each was built from about 300,000 clones for pCTRL2 and 270,000 clones for pCTRL2.T7.100.sRBS.
3. Co-transformation of G-GECO1 with the library and control reference in chemically competent BL21(DE3)
[0295] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.sRBS-.sup.3×NNKS.sup.2168
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2-.sup.3×NNKS.sup.2168
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.sRBS-S.sup.2171 M4A
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2-S.sup.2171 M4A
4. Inoculation Overnight:
[0296] Colonies of BL21(DE3) on LB agar from 3. were picked and cultivated in deep well plates with 300 μl of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 1300 rpm.
5. Microtiter Plate Readout:
[0297] After cultivating individual clones from 4. overnight, the OD.sub.600 of the precultures in microtiter plates was adjusted to 0.1, and the samples, 200 μL each, were shaken at 37° C. and 180 rpm for 30 min. Within the framework of the pCTRL2-.sup.3×NNKS.sup.2168 library, 271 clones were thus picked. Within the framework of the pCTRL2.T7.100.sRBS-.sup.3×NNKS.sup.2168 library, 576 clones were picked.
[0298] Thereafter, the G-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, and the fluorescence was measured over 110 min.
[0299] Subsequently, all nanopore variants were then induced with 0.5 mM IPTG, and the fluorescence was measured over another 190 min.
[0300] General information: The measurement in the reader is carried out at 30° C. every 3 min. The plate is shaken at 180 rpm between the measurements. The OD.sub.600 and the fluorescence (Ex: 485 nm; Em: 525 nm) are measured. The volume in the cavities is 200 μl after addition of all inductors.
6. Analysis
[0301] For the analysis, the curves were fitted empirically with equation 1 in order to quantitatively determine the half-maximum time c or the slope d, for example. The distribution of the parameters investigated with respect to the half-maximum time c and the slope d of the two libraries pCTRL2-.sup.3×NNKS.sup.2168 and pCTRL2.T7.100.sRBS-.sup.3×NNKS.sup.2168 is summarized in
[0302] Individual mutants from the pCTRL2.T7.100.sRBS-.sup.3×NNKS.sup.2168 library were subsequently sequenced (240 sequenced of the total of 576 measured) and subjected to a detailed sequence structure function analysis. Sequences with methionine were not taken into account here due to the possibility of an alternatively start codon. Sequences with premature stop codons were also not taken into account. Sequences with cysteines were also not taken into account due to possible disulfide bridges. The same applies to prolines, which only rarely occurred. In addition, sequences with reading-frame shift were also not taken into account. This ultimately resulted in a selection of 142 sequences of originally 240 sequenced variants, which were respectively analyzed.
[0303] For the analysis of the underlying structure-function properties, the average half-maximum time c and the average slope d were calculated in each case for each individual amino acid at the three different positions X.sub.1, x.sub.2 and X.sub.3 (
[0304] In parallel, the individual amino acids were combined into groups with comparable chemical properties, and the average half-maximum time c and the average slope d were calculated in each case. These results are summarized as a heat map (
[0305] In addition, specific sequences with characteristic pore-forming properties (in each case positive and negative) together with the values for the respective half-maximum time c and the slope d are summarized in Table 1.
Conclusion:
[0306] In addition to Example 4, within the scope of a screening campaign with up to 8,000 randomized variants, the influence of the N-terminal sequence of the S.sup.21-71 pinholin on its pore-forming properties was systematically investigated.
[0307] Initially, with the aid of the weaker promoters (specifically, T7.100.sRBS in contrast to T7.wt.sRBS), the pore-forming properties of the .sup.3×NNKS.sup.2168 library were better experimentally resolved than with the stronger promoter (
[0308] In addition, a sequence-structure-function analysis shows that negative charges at position 3 measured at the half-maximum time c accelerate pore formation by way of example (
TABLE-US-00025 TABLE 1 Singularized clones from the screening of the .sup.3xNNKS21-68 pinholin library N-terminal Half-maximum Clone ID sequence time [c] Slope [d] Comment P14E5 YLE 166.1 4.908 Accelerating P14C11 NFE 172.3 6.835 Accelerating P15D3 DWD 187.4 6.727 Accelerating P7D10 AGW 304.1 22.49 Inhibitory P3G3 WRG 358.7 47.62 Inhibitory P3E5 FQW 483.1 28.25 Inhibitory Note: The complete sequences of the following clones are summarized in the sequence listing under SEQ ID NOs: 44-49.
Example 13—Selection Experiments for Finding New Properties and Functions of Pore-Forming Membrane Peptides in Microtiter Plates III
Background:
[0309] In addition to the model selections (see Example 3) and the screening of the S.sup.2168 pinholin library with up to 8,000 different variants (see Example 4 and 12), the S.sup.21 68 pinholin was additionally truncated in increments of two amino acids and subjected to a functional investigations with the aid of the method.
[0310] On the one hand, there must be a check of the extent to which conclusive structure-function properties can be identified with the aid of the method in the context of a systematic screening. On the other hand, there must be a check of which components are sufficient and necessary for the pore formation of the S.sup.2168 pinholin. Such minimal nanopore motifs then offer, by way of example, promising starting points for further construction projects. In addition, there must be a check of the extent to which course of the fluorescent signal is comparable for different screening formats, specifically in microtiter plates and on the basis of individual colonies on agar plates.
Experimental Procedure and Results:
Variant 1: Screening of the S.SUP.21.68 Truncation Library in Microtiter Plates
[0311] 1. Co-Transformation of G-GECO1 and the Truncated S.sup.2168 Pinholin (SEQ ID NOs: 28-42) in Chemically Competent E. coli.
[0312] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.sRBS-S.sup.2168 truncation library
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.sRBS-S.sup.2171
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.sRBS-S.sup.2171 M4A
2. Inoculation Overnight:
[0313] Colonies of BL21(DE3) on LB agar from 1. were picked and cultivated in deep well plates with 300 μl of LB medium with the necessary antibiotics overnight for about 16 h at 37° C. and 1300 rpm.
3. Microtiter Plate Readout:
[0314] After cultivating individual clones from 2. overnight, the OD.sub.600 of the precultures in microtiter plates was adjusted to 0.1, and the samples, 200 μL each, were shaken at 37° C. and 180 rpm for 30 min.
[0315] Thereafter, the G-GECO1 was initially induced with 25 mM sodium propionate, pH 8.0, and the course of the fluorescent signal was measured over 110 min.
[0316] Subsequently, the expression of the S.sup.2168 truncation variant was then induced with 0.5 mM IPTG, and the course of the fluorescent signal was measured over another 190 min.
[0317] General information: The measurement in the microtiter plate spectrometer was carried out at 30° C. at intervals of 3 min. The microtiter was shaken at 180 rpm between the measurements. The OD.sub.600 and the fluorescence (Ex: 485 nm; Em: 525 nm) are measured. The volume in the cavities is 200 μl after addition of all inductors.
4. Experimental Analysis:
[0318] For the analysis, the curves were fitted empirically with equation 1 in order to quantitatively determine the half-maximum time c or the slope d. The distribution of the parameters investigated with respect to the half-maximum time c and the slope d of the truncation is summarized in (
Variant 2: Screening of the S.SUP.21.68 Truncation Library in Agar Plates
[0319] 1. Co-Transformation of G-GECO1 and the Truncated S.sup.2168 Pinholin (SEQ ID NOs: 28-42) in Chemically Competent E. coli.
[0320] Transformed individually in each case:
.fwdarw.BL21(DE3) with pPRO24-G-GECO1 (reporter)+pCTRL2.T7.100.sRBS-S.sup.2168 truncation library.
2. Growth Overnight:
[0321] Colonies of BL21(DE3) were spread onto LB agar and grown overnight for about 16 hours at 37° C.
3. Agar Plate Readout:
[0322] At the beginning, the reporter G-GECO1 was induced by spraying 500 mM sodium propionate, pH 8.0, onto the agar plate using an airbrush, so that all colonies were slightly wetted. The agar plates were then incubated in the dark at 20° C. for 180 minutes.
[0323] Thereafter, the expression of the truncation variants was induced by spraying 50 mM IPTG solution onto the agar plate, again using an airbrush and slightly wetting all colonies. The agar plates were then incubated in the dark at 20° C. for another 240 minutes.
[0324] From the time of IPTG addition, images were recorded at a distance of one 15 hour starting at the time 0 minutes to 240 minutes
[0325] General information: The measurement is carried out in blue light in a gel documentation device (Ex: 470 nm; Em: F-590.M58 UV filter). All plates were recorded with the same exposure time to guarantee comparability.
4. Experimental Analysis:
[0326] For the analysis, the data of one image detail each were analyzed with the aid of image analysis software (Fiji ImageJ 1.51; Nature Methods, 2012, 9, 676-82), and the maximum fluorescence was plotted against the time (
Conclusion:
[0327] Specifically, a truncation of the S.sup.2-68 pinholin in increments of two 30 amino acids causes different fluorescent signals and thereby provide new insights into the structure-function properties, which are the basis for the pore formation of the S.sup.21-68 pinholin. Measured at the half-maximum time c and the slope d, the two N-terminal amino acids D and K in the context of S.sup.21-68 pinholin have an inhibitory effect on the formation of the nanopore. A further truncation improves the pore-forming properties until the complete truncation of the aromatic motif YWFQLW. By way of example, the S.sup.2-48 pinholin variant can now be used as a minimal nanopore motif for further construction projects.
[0328] With regard to the different screening formats, the course of the fluorescent signals in microtiter plates (quantified and summarized in