Polypeptide mixes with antibacterial activity
09789167 · 2017-10-17
Assignee
Inventors
Cpc classification
C12N9/00
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C12N9/80
CHEMISTRY; METALLURGY
A61K38/50
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A61K38/54
HUMAN NECESSITIES
International classification
C12N9/80
CHEMISTRY; METALLURGY
C12N9/50
CHEMISTRY; METALLURGY
A61K38/54
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
C12N9/00
CHEMISTRY; METALLURGY
A61K38/16
HUMAN NECESSITIES
Abstract
The invention relates to the field of microbiology, specifically to a combination of a source of a first enzymatic active domain and a source of a second enzymatic active domain and to a composition comprising said combination. The invention further relates to a composition comprising said combination for use as a medicament, to the use of said composition as an antimicrobial agent and to a method for controlling microbial contamination in a food- or feed product, on and/or in food- or feed processing equipment, on and/or in food- or feed containers.
Claims
1. A composition comprising a combination of a source of a first enzymatic active domain and a source of a second enzymatic active domain, wherein said source comprises a polypeptide, wherein said first and second enzymatic active domains each exhibit distinct target bond specificities and are comprised on a distinct first and second polypeptide, and wherein said first and second polypeptide comprise a different number of copies of said first and/or second enzymatic active domain, and wherein said first polypeptide comprises a sequence that has at least 80% sequence identity to SEQ ID NO: 58, said second polypeptide comprises a sequence that has at least 80% sequence identity to SEQ ID NO: 70.
2. A composition according to claim 1, wherein said different target bonds are essential bonds in a peptidoglycan layer of a bacterial cell, wherein said bacterial cell is a Staphylococcus.
3. The composition according to claim 1, wherein: said combination further comprises a source of a third enzymatic active domain comprised on a distinct third polypeptide, said third enzymatic active domain is an amidase domain, said distinct third polypeptide further comprises a cell wall-binding domain, and each of said distinct first, second and third polypeptide comprises a different number of copies of said first, second and third enzymatic active domain.
4. The composition according to claim 3, wherein said third polypeptide comprises a sequence that has at least 80% sequence identity SEQ ID NO: 52.
5. The composition according to claim 1, further comprising an pharmaceutical acceptable carrier and/or an additional active ingredient selected from the group consisting of a bacteriophage, a bacteriostatic agent, a bactericide agent, an antibiotic, a surfactant and/or an enzyme.
6. A composition comprising a combination of a source of a first enzymatic active domain, a source of a second enzymatic active domain, and a source of a third enzymatic domain, wherein said source comprises a polypeptide; wherein each of said first, second and third enzymatic active domains exhibits distinct target bond specificities and are comprised of distinct first, second and third polypeptides and each of said distinct first, second and third polypeptides comprises a different number of copies of said first, second and third enzymatic active domains, and further comprises a cell wall-binding domain; wherein said first enzymatic active domain is a cysteine, histidine-dependent amidohydrolases/peptidase domain, said second enzymatic active domain is an endopeptidase domain, said third enzymatic domain is an amidase domain; and wherein said first polypeptide comprises a sequence that has at least 80% sequence identity to SEQ ID NO: 58, said second polypeptide comprises a sequence that has at least 80% sequence identity SEQ ID NO: 70 and said third polypeptide comprises a sequence that has at least 80% sequence identity SEQ ID NO: 52.
7. A method of treatment, prevention or delay of a Staphylococcus related condition in an individual, comprising administering to said individual a composition according to claim 1.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
EXAMPLES
Example 1
(21) Materials and Methods
(22) Bacteria, Phages, and Plasmids
(23) Bacterial strains for cloning and protein production, phages, and plasmids used in this study are listed in Table 1. E. coli XL1-Blue MRF′ (Stratagene, La Jolla, Calif., U.S.) and E. coli Sure (Stratagene) served for cloning and over-expression of N-terminal 6xHis-tagged recombinant fusion proteins. Constructs containing repetitive sequences were processed in E. coli Sure strain. E. coli was cultured in Luria-Bertani (LB) medium at 37° C. supplemented with 100 μg/ml ampicillin and 30 μg/ml tetracycline for cloning, and at 30° C. with 100 μg/ml ampicillin for plasmid selection during protein expression. Staphylococcus aureus, BB270 NCTC8325mec used as substrate in lysis assays, was grown in, half concentrated Brain Heart Infusion medium (BHI, Biolife, Milano, Italy) at 37° C. Log phase cells from two liter cultures were harvested, PBST (50 mM NaH.sub.2PO.sub.4, 120 mM NaCl, 0.1% Tween 20, pH 7.4) washed, 100 fold concentrated and aliquots thereof were stored at −80° C.
(24) DNA Techniques and Cloning Procedures
(25) Cloning and construction of fusion proteins were performed using standard techniques (Loessner et al. Mol Microbiol 2002, 44: 335-349; Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York). Enzymes were purchased from New England Biolabs (Ipswitch, Mass., U.S.), Fermentas (Burlington, Canada, Roche Basel, Switzerland) and Qiagen. Endolysins and separated enzymatically active domains (EAD) coding regions from phages Φ2638a, Φ187, ΦK, and ΦTwort were in frame amplified from purified phage DNA or phage lysate. Plasmid DNA served as template for amplification of EAD encoding gene fragments of lysostaphin with High Fidelity PCR Enzyme Mix (Fermentas). Restriction sites for insert ligation into pQE-30 protein expression plasmid (Qiagen) and its derivatives were introduced by the primers. Plasmids constructed or used in this study are listed in Table 1. Protein expression plasmids were transformed into electro-competent E. coli XL1BlueMRF and Plasmids containing repetitive sequences into electro-competent E. coli Sure. DNA concentrations were determined with a spectrophotometer (NanoDrop ND-1000 Spectrophotometer, Thermo scientific, Waltham, Mass., U.S.). Sequence integrities were confirmed by nucleotide sequencing (GATC, Konstanz, Germany). Constructs bearing a single N-terminal enzymatically active domain (EAD) and a C-terminal cell wall binding domain (CBD), or a cell wall targeting domain (CWT) respectively were constructed by creating in-frame fusions of the respective coding regions with splicing overlap extension PCR (SOE). These fragments were inserted into SacI-SalI restriction sites of pQE-30Xa vector DNA. On the basis of these vectors, constructs with repetitive duplicated EADs were obtained by introducing the respective EAD coding sequences into StuI-SacI sites. For full construction principles please refer to Table 1.
(26) Expression and Purification of Recombinant Fusion-Proteins
(27) Protein overexpression and immobilized metal affinity chromatography (IMAC) purification of N-terminal 6xHis tagged proteins was done with minor modifications as previously described by others (Loessner et al. Appl Environ Microbiol 1996, 62: 3057-3060; Schmelcher et al. Appl Environ Microbiol 2010, 76: 5745-5756; Eichenseher et al., unpublished). Briefly, heterologous proteins expression was induced by the addition of 0.2-0.5 mM IPTG to log phase E. coli cultures, grown in LB medium at 30° C. Cells were further incubated at the same temperature before harvesting by centrifugation. E. coli were lysed in immobilization buffer (50 mM NaH.sub.2PO.sub.4, 500 mM NaCl, 5 mM imidazole, 0.1% Tween 20, pH 7.4) by a double passage through a French Pressure Cell Press (1200 psi, SLM Aminco, Urbana, Ill., U.S.) operated at 1200 psi. Insoluble cell debris was removed by centrifugation and filter sterilization (0.2 μm PES membrane, Millipore) prior to gravity flow IMAC purification in MicroBiospin (Bio-Rad, Hercules, Calif., U.S.) columns packed with low density Ni-NTA Superflow resin (Chemie Brunschwig AG, Basel, Switzerland). After column washing, 6xHis tagged proteins were eluted using elution buffer (50 mM NaH.sub.2PO.sub.4, 500 mM NaCl, 125 mM imidazole, 0.1% Tween 20, pH 7.4) and dialyzed against dialysis buffer (50 mM NaH.sub.2PO.sub.4, 100 mM NaCl, 0.1% Tween 20, pH 7.4). CHAP homologues domain containing proteins were subjected to buffer exchange using EconoPak 10DG columns (Biorad) using CHAP buffer (50 mM Tris, 5 mM CaCl.sub.2, 10% glycerol, pH 7.4). Protein purities were estimated by SDS-PAGE and concentrations were determined spectrophotometrically (NanoDrop ND-1000 spectrophotmeter) or with a Pierce BCA Protein Assay Kit (Thermo Fischer Scientific, Waltham, Mass., U.S.) according to the manufacturer s manual. Proteins were stored in 50% glycerol at −20° C.
(28) Photometric Determination of Lysis Kinetics
(29) Lytic activities were measured in turbidity reduction assays using a Wallac VICTOR.sup.3 TM14200 (Perkin Elmer, Waltham, Mass., U.S.) multilable counter device. Substrate cells from frozen stock were washed with buffer and adjusted to an optical density at 595 nm (OD.sub.595 nm) of 1+/−0.05 using Macro Cuvettes (Greiner Bio-one, Kremsmünster, Austria) and a spectrophotometer (BioChrom, Cambridge, UK). Staphylococcus lytic enzymes were diluted with buffer to equimolar quantities and if desired, subsequently pooled to obtain enzyme mixtures. 10 μl protein solutions were distributed in crystal grade multi-well polystyrene tissue culture test plates (SPL Lifesciences, Poncheon-Si, Korea) and mixed with 190 μl substrate cell suspension using a multichannel pipette. Reduction in turbidity over the time was monitored at OD.sub.595 nm with vigorous plate shaking in between the reads. As a control to monitor autolytic activity under the given conditions served 10 μl buffer or water. Assays were performed in triplicates. Calculation of relative activity values were obtained as described elsewhere (Korndorfer et al. J Mol Biol 2006, 364: 678-689; Schmelcher et al., Microb Biotechnol. 2011, 4(5): 651-662). Sigmoidal lysis- and control curves were normalized to a common starting value of 1.
(30) Results
(31) Downstream processing of cytosolic expressed Staphylococcus lytic proteins resulted in soluble proteins with purities depending on the protein structure and origin. The majority of the constructs had by SDS-PAGE estimated purities of up to >90% (
(32) We tested a selection of the partially purified proteins in turbidity reduction assays (lysis assays). Individual lysins and combinations thereof were tested against S. aureus BB270 cells from frozen stock in PBST buffer at pH 7.4 and at different protein concentrations. CHAPK_CHAPK_CWT-LST (SEQ ID NO: 58, encoded by SEQ ID NO: 47) and CHAPK_CHAPK_CBD2638 (SEQ ID NO: 56 encoded by SEQ ID NO: 55) proteins were virtually inactive at 50 nM assay concentrations against a cell suspension set to an optical density at 595 nm (OD595 nm) of ˜1, but displayed significant activities at 200 nM assay concentrations (
(33) Using M23-LST (SEQ ID NO: 16, encoded by SEQ ID NO: 15) containing proteins in an identical assay setup, best results were achieved using M23-LST_M23-LST_CWT-LST (SEQ ID NO: 70, encoded by SEQ ID NO: 69) at 200 nM assay concentration. The CWT-LST (SEQ ID NO: 4, encoded by SEQ ID NO: 3) appears to be superior to CBD2638 (SEQ ID NO: 6, encoded by SEQ ID NO: 5). Furthermore, repetitive double M23-LST variants (SEQ ID NO: 68 and 70 encoded by SEQ ID NO: 67 and 69, respectively) were found superior to single M23-LST (SEQ ID NO: 44 and 46, encoded by SEQ ID NO: 43 and 45, respectively). This effect was found more pronounced at 50 nM protein concentrations. For full results please refer to
(34) All lysins built with Ami2638 (SEQ ID NO: 18, encoded by SEQ ID NO: 17) were significantly less active compared to CHAPK (SEQ ID NO: 10, encoded by SEQ ID NO: 9) and M23-LST (SEQ ID NO: 16, encoded by SEQ ID NO: 15) proteins. Here, CBD2638 (SEQ ID NO: 6 encoded by SEQ ID NO: 5) was superior to CWT-LST (SEQ ID NO: 4, encoded by SEQ ID NO: 3). Duplication of the catalytic domain had little effect when combined with CBD2638 (SEQ ID NO: 6, encoded by SEQ ID NO: 5), but duplication added a positive effect on lysis kinetics when combined with CWT-LST (SEQ ID NO: 4, encoded by SEQ ID NO: 3) (
(35) We also compared activities of mixtures of proteins built with CWT-LST (SEQ ID NO: 4, encoded by SEQ ID NO: 3) or CBD2638 (SEQ ID NO: 6, encoded by SEQ ID NO: 5). At low protein concentrations (16.67 nM each, or 50 nM total protein concentration respectively), mixtures of CWT-LST (SEQ ID NO: 58, 70 and 52) proteins were found significantly more active than mixtures of CBD2638 (SEQ ID NO: 56, 68 and 50) proteins. Furthermore, duplication of the EADs had little effect on lysis kinetics in CBD2638 constructs mixtures (SEQ ID NO: 56, 68 and 50 as compared to SEQ ID NO: 32, 44 and 26) (
(36) Finally, we compared the most effective mixture consisting of Ami2638_Ami2638_CWT-LST (SEQ ID NO: 52, encoded by SEQ ID NO: 51), CHAPK_CHAPK_CWT-LST (SEQ ID NO: 58, encoded by SEQ ID NO: 57), and M23-LST_M23-LST_CWT-LST (SEQ ID NO: 70, encoded by SEQ ID NO: 69) with the most effective reference protein M23-LST_M23-LST_CWT-LST (SEQ ID NO: 70, encoded by SEQ ID NO: 69). At both concentrations tested (50 nM and 200 nM total protein concentrations), the mixtures were found slightly superior to M23-LST_M23-LST_CWT-LST (SEQ ID NO: 70, encoded by SEQ ID NO: 69) (
Example 2
Material and Methods
(37) The lysis kinetics of single and combinations/mixtures of protein constructs produced according to Example 1 have been tested using the turbidity reduction assay as described in the Material and Method section of Example 1.
(38) Results
(39) Lysis curves of the proteins and mixtures are shown in
(40) TABLE-US-00001 TABLE 1 Bacterial strains, bacteriophages and plasmids Source or Strain, phage, or plasmid Genotype or relevant characteristics reference Bacterial strains E. coli XL-1BlueMRF' Δ mcrA 183 Δ mcrCB-hsdSMR-mrr 173 endA1 Stratagene supE44 thi-1 recA1 gyrA96 relA1 lac[F' proAB laclq ZΔM15 Tn10 Tet.sup.r] E. coli Sure e14- McrA- Δ mcrCB-hsdSMR-mrr 171 endA1 Stratagene supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 Kan.sup.r uvrC [F' proAB laclqZΔM15 Tn10 Tet.sup.r] Phages S. aureus Φ187 Siphoviridae Loessner et al. J Bacteriol 1999, 181: 4452-4460. S. aureus Φ2638 Siphoviridae Kwan et al. Proc Natl Acad Sci U S A 2005, 102: 5174-5179. S. aureus ΦK Siphoviridae O'Flaherty et al. J Bacteriol 2005, 187: 7161-7164. S. aureus ΦTw Siphoviridae Loessner et al. FEMS Microbiol Lett 1998, 162: 265-274. Plasmids pQE-30Xa 3.4 kb cloning and expression vector; T5 Qiagen promoter; creates N-terminal fusion of gene product with 21-aminoacid leader containing a 6x His-tag and Factor Xa protease cleavage site; Amp.sup.r pHXaAm2638_CBD2638 ply2638 fragment encoding Ala143 - Lys486 This study cloned into SacI - SalI sites of pQE-30Xa pHXaAm2638_CWT-LST In-frame fusions of ply2638 fragment encoding This study Ala143 - Asp392 and pre- pro- lysostaphin encoding Trp402 - Lys493 cloned into SacI - SalI sites of pQE-30Xa pHXaAm2638_CWT-NM3 In-frame fusions of ply2638 fragment encoding This study Ala143 - Asp392 and ply187 encoding Gly158 - Phe251 cloned into SacI - SalI sites of pQE- 30Xa pHXaCHAPK_CBD2638 In-frame fusions of lysK fragment encoding Met1 - This study Ala165 and ply2638 encoding Gly360 - Lys486 cloned into SacI - SalI sites of pQE- 30Xa pHXaCHAPK_CWT-LST In-frame fusions of lysK fragment encoding Met1 - This study Ala165 and pre-pro-lysostaphin encoding Gly388 - Lys493 cloned into SacI - SalI sites of pQE-30Xa pHXaCHAPK_CWT-NM3 In-frame fusions of lysK fragment encoding Met1 - This study Ala165 and ply187 encoding Gly158 - Phe251 cloned into SacI - SalI sites of pQE-30Xa pHXaCHAPTw_CBD2638 In-frame fusions of PlyTw fragment encoding This study Met1 - Asn182 and ply2638 encoding Trp393 - Lys486 cloned into SacI - SalI sites of pQE- 30Xa pHXaCHAPTw_CWT-LST In-frame fusions of PlyTw fragment encoding This study Met1 - Asn182 and pre-pro-lysostaphin encoding Trp402 - Lys493 cloned into SacI - SalI sites of pQE-30Xa pHXaCHAPTw_CWT- In-frame fusions of PlyTw fragment encoding This study NM3 Met1 - Ala165 and ply187 encoding Gly158 - Phe251 cloned into SacI - SalI sites of pQE- 30Xa pHXaM23-LST_CBD2638 In-frame fusions of pre- pro- lysostaphin This study fragment encoding Ala251 - Pro398 and ply2638 encoding Trp393 - Lys486 cloned into SacI - SalI sites of pQE-30Xa pHXaM23-LST_CWT-LST pre- pro- lysostaphin fragment encoding Ala251 - This study Lys493 cloned into SacI - SalI sites of pQE- 30Xa pHXaM23-LST_CWT- In-frame fusions of pre- pro- lysostaphin This study NM3 fragment encoding Ala251 - Gly401 and ply187 encoding Gly158 - Phe251 cloned into SacI - SalI sites of pQE-30Xa pHXaAm2638_Am2638_ ply2638 fragment encoding Ala143 - Gly359 This study CBD2638 cloned into StuI - SacI sites of pHXaAm2638_CBD2638 pHXaAm2638_Am2638_ ply2638 fragment encoding Ala143 - Gly359 This study CWT-LST cloned into StuI - SacI sites of pHXaAm2638_CWT-LST pHXaAm2638_Am2638_ ply2638 fragment encoding Ala143 - Gly359 This study CWT-NM3 cloned into StuI - SacI sites of pHXaAm2638_CWT-NM3 pHXaCHAPK_CHAPK_ lysK fragment encoding Met1 - Asn195 cloned This study CBD2638 into StuI - SacI sites of pHXaCHAPK_CBD2638 pHXaCHAPK_CHAPK_ lysK fragment encoding Met1 - Asn195 cloned This study CWT-LST into StuI - SacI sites of pHXaCHAPK_CWT-LST pHXaCHAPK_CHAPK_ lysK fragment encoding Met1 - Asn195 cloned This study CWT-NM3 into StuI - SacI sites of pHXaCHAPK_CWT-NM3 pHXaCHAPTw_CHAPTw_ plyTw fragment encoding Met1 - Asn182 cloned This study CBD2638 into StuI - SacI sites of pHXaCHAPTw_CBD2638 pHXaCHAPTw_CHAPTw_ plyTw fragment encoding Met1 - Asn182 cloned This study CWT-LST into StuI - SacI sites of pHXaCHAPTw_CWT- LST pHXaCHAPTw_CHAPTw_ plyTw fragment encoding Met1 - Asn182 cloned This study CWT-NM3 into StuI - SacI sites of pHXaCHAPTw_CWT- NM3 pHXaM23-LST_ pre- pro- lysostaphin fragment encoding Ala251 - Donovan et al. M23-LST_CBD2638 Gly401 cloned into StuI - SacI sites of FEMS Microbiol pHXaM23-LST_CBD2638 Lett 2006, 265: 133-139. pHXaM23-LST_ pre- pro- lysostaphin fragment encoding Ala251 - This study M23-LST_CWT-LST Gly401 cloned into StuI - SacI sites of pHXaM23-LST_CWT-LST pHXaM23-LST_ pre- pro- lysostaphin fragment encoding Ala251 - This study M23-LST_CWT-NM3 Gly401 cloned into StuI - SacI sites of pHXaM23-LST_CWT-NM3
(41) TABLE-US-00002 TABLE 2 SEQ ID NO overview table SEQ ID NO Name construct organism 1 Ply2638 endolysin CDS Bacteriophage 2638A 2 Ply2638 endolysin PRT Bacteriophage 2638A 3 CWT-LST CDS S. simulans 4 CWT-LST PRT S. simulans 5 CBD2638 CDS Bacteriophage 2638A 6 CBD2638 PRT Bacteriophage 2638A 7 CWT-NM3 CDS S. aureus phage phiNM3 8 CWT-NM3 PRT S. aureus phage phiNM3 9 CHAPK CDS S. phage K 10 CHAPK PRT S. phage K 11 CHAP-φTwort CDS S. phage Twort 12 CHAP-φTwort PRT S. phage Twort 13 M23-2638 CDS Bacteriophage 2638A 14 M23-2638 PRT Bacteriophage 2638A 15 M23-LST CDS S. simulans 16 M23-LST PRT S. simulans 17 Ami2638 CDS Bacteriophage 2638A 18 Ami2638 PRT Bacteriophage 2638A 19 CHAPK_CHAPK_CWT-LST CDS artificial construct 20 CHAPK_CHAPK_CWT-LST PRT artificial construct 21 M23-LST_M23-LST_CWT-LST CDS artificial construct 22 M23-LST_M23-LST_CWT-LST PRT artificial construct 23 Ami2638_ami2638_CWT-LST CDS artificial construct 24 Ami2638_ami2638_CWT-LST PRT artificial construct 25 HXaAmi2638_CBD2638 CDS artificial construct 26 HXaAmi2638_CBD2638 PRT artificial construct 27 HXaAmi2638_CWT-LST CDS artificial construct 28 HXaAmi2638_CWT-LST PRT artificial construct 29 HXaAmi2638_CWT-NM3 CDS artificial construct 30 HXaAmi2638_CWT-NM3 PRT artificial construct 31 HXaCHAPK_CBD2638 CDS artificial construct 32 HXaCHAPK_CBD2638 PRT artificial construct 33 HXaCHAPK_CWT-LST CDS artificial construct 34 HXaCHAPK_CWT-LST PRT artificial construct 35 HXaCHAPK_CWT-NM3 CDS artificial construct 36 HXaCHAPK_CWT-NM3 PRT artificial construct 37 HXaCHAPTw_CBD2638 CDS artificial construct 38 HXaCHAPTw_CBD2638 PRT artificial construct 39 HXaCHAPTw_CWT-LST CDS artificial construct 40 HXaCHAPTw_CWT-LST PRT artificial construct 41 HXaCHAPTw_CWT-NM3 CDS artificial construct 42 HXaCHAPTw_CWT-NM3 PRT artificial construct 43 HXaM23-LST_CBD2638 CDS artificial construct 44 HXaM23-LST_CBD2638 PRT artificial construct 45 HXaM23-LST_CWT-LST CDS artificial construct 46 HXaM23-LST_CWT-LST PRT artificial construct 47 HXaM23-LST_CWT-NM3 CDS artificial construct 48 HXaM23-LST_CWT-NM3 PRT artificial construct 49 HXaAmi2638_Ami2638_CBD2638 CDS artificial construct 50 HXaAmi2638_Ami2638_CBD2638 PRT artificial construct 51 HXaAmi2638_Ami2638_CWT-LST CDS artificial construct 52 HXaAmi2638_Ami2638_CWT-LST PRT artificial construct 53 HXaAmi2638_Ami2638_CWT-NM3 artificial construct CDS 54 HXaAmi2638_Ami2638_CWT-NM3 artificial construct PRT 55 HXaCHAPK_CHAPK_CBD2638 CDS artificial construct 56 HXaCHAPK_CHAPK_CBD2638 PRT artificial construct 57 HXaCHAPK_CHAPK_CWT-LST CDS artificial construct 58 HXaCHAPK_CHAPK_CWT-LST PRT artificial construct 59 HXaCHAPK_CHAPK_CWT-NM3 CDS artificial construct 60 HXaCHAPK_CHAPK_CWT-NM3 PRT artificial construct 61 HXaCHAPTw_CHAPTw_CBD2638 CDS artificial construct 62 HXaCHAPTw_CHAPTw_CBD2638 PRT artificial construct 63 HXaCHAPTw_CHAPTw_CWT-LST artificial construct CDS 64 HXaCHAPTw_CHAPTw_CWT-LST artificial construct PRT 65 HXaCHAPTw_CHAPTw_CWT-NM3 artificial construct CDS 66 HXaCHAPTw_CHAPTw_CWT-NM3 artificial construct PRT 67 HXaM23-LST_M23-LST_CBD2638 artificial construct CDS 68 HXaM23-LST_M23-LST_CBD2638 artificial construct PRT 69 HXaM23-LST_M23-LST_CWT-LST artificial construct CDS 70 HXaM23-LST_M23-LST_CWT-LST artificial construct PRT 71 HXaM23-LST_M23-LST_CWT-NM3 artificial construct CDS 72 HXaM23-LST_M23-LST_CWT-NM3 artificial construct PRT 73 His-tag with linker CDS artificial construct 74 His-tag with linker PRT artificial construct
(42) TABLE-US-00003 TABLE 3 Results of Example 2. protein construct SEQ ID NO mean (Δ.sub.maxOD.sub.595 nm*min.sup.−1 std.dev. std.err. Single protein constructs (30 nM protein concentration) HXaAmi2638_CWT-LST 28 −0.004345 0.000722 0.000511 HXaCHAPK_CWT-LST 34 −0.005163 0.000552 0.000390 HXaM23-LST_CWT-LST 46 −0.013310 0.000387 0.000224 HXaAmi2638_Ami2638_CWT-LST 52 −0.006686 0.000462 0.000267 HXaCHAPK_CHAPK_CWT-LST 58 −0.004086 0.000304 0.000176 HXaM23-LST_M23-LST_CWT-LST 70 −0.040422 0.000624 0.000360 Mixtures of two protein constructs (30 nM total, 15 nM each protein) HXaAmi2638_CWT-LST 28 + 34 −0.004492 0.000222 0.000157 HXaCHAPK_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 58 −0.010524 0.002527 0.001459 HXaCHAPK_CHAPK_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 34 −0.004471 0.000125 0.000072 HXaCHAPK_CWT-LST HXaAmi2638_CWT-LST 28 + 58 −0.006872 0.000850 0.000491 HXaCHAPK_CHAPK_CWT-LST HXaAmi2638_CWT-LST 28 + 46 −0.018363 0.000199 0.000115 HXaM23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 70 −0.060616 0.004117 0.002377 HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 46 −0.020094 0.001989 0.001148 HXaM23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 70 −0.049715 0.005762 0.003327 HXaM23-LST_M23-LST_CWT-LST HXaCHAPK_CWT-LST 34 + 46 −0.009839 0.000700 0.000404 HXaM23-LST_CWT-LST HXaCHAPK_CHAPK_CWT-LST 58 + 70 −0.039957 0.001111 0.000641 HXaM23-LST_M23-LST_CWT-LST HXaCHAPK_CHAPK_CWT-LST 58 + 46 −0.011577 0.003904 0.002254 HXaM23-LST_CWT-LST HXaCHAPK_CWT-LST 34 + 70 −0.029355 0.000913 0.000527 HXaM23-LST_M23-LST_CWT-LST Mixtures of three protein constructs (30 nM total, 10 nM each protein) HXaAmi2638_CWT-LST 28 + 34 + 46 −0.013973 0.001444 0.000834 HXaCHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 58 + 70 −0.052270 0.007606 0.004391 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 58 + 70 −0.045011 0.003443 0.001988 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 34 + 70 −0.042337 0.003308 0.001910 HXaCHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 58 + 46 −0.020569 0.003307 0.001910 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 34 + 46 −0.016268 0.000576 0.000333 HXaCHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 58 + 46 −0.013975 0.000365 0.000211 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 34 + 70 −0.036804 0.003481 0.002010 HXaCHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST Single protein constructs (50 nM proteinconcentration) HXaAmi2638_CWT-LST 28 −0.005109 0.000061 0.000035 HXaCHAPK_CWT-LST 34 −0.004037 0.000369 0.000261 HXaM23-LST_CWT-LST 46 −0.022770 0.000304 0.000175 HXaAmi2638_Ami2638_CWT-LST 52 −0.008042 0.000593 0.000342 HXaCHAPK_CHAPK_CWT-LST 58 −0.003674 0.000121 0.000086 HXaM23-LST_M23-LST_CWT-LST 70 −0.054314 0.000820 0.000474 Mixtures of two protein constructs (50 nM total, 25 nM each protein) HXaAmi2638_CWT-LST 28 + 34 −0.004611 0.000945 0.000668 HXaCHAPK_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 58 −0.007071 0.000287 0.000203 HXaCHAPK_CHAPK_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 34 −0.006845 0.000578 0.000334 HXaCHAPK_CWT-LST HXaAmi2638_CWT-LST 28 + 58 −0.005107 0.000756 0.000436 HXaCHAPK_CHAPK_CWT-LST HXaAmi2638_CWT-LST 28 + 46 −0.029038 0.000591 0.000341 HXaM23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 70 −0.077677 0.005683 0.003281 HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 46 −0.033351 0.001808 0.001044 HXaM23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 70 −0.076113 0.000463 0.000268 HXaM23-LST_M23-LST_CWT-LST HXaCHAPK_CWT-LST 34 + 46 −0.014407 0.000917 0.000529 HXaM23-LST_CWT-LST HXaCHAPK_CHAPK_CWT-LST 58 + 70 −0.048809 0.000527 0.000304 HXaM23-LST_M23-LST_CWT-LST HXaCHAPK_CHAPK_CWT-LST 58 + 46 −0.018130 0.001014 0.000586 HXaM23-LST_CWT-LST HXaCHAPK_CWT-LST 34 + 70 −0.046676 0.002135 0.001233 HXaM23-LST_M23-LST_CWT-LST Mixtures of three protein constructs (50 nM total, 16.67 nM each protein) HXaAmi2638_CWT-LST 28 + 34 + 46 −0.020491 0.001630 0.000941 HXaCHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 58 + 70 −0.062127 0.002998 0.001731 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 58 + 70 −0.054493 0.002078 0.001200 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 34 + 70 −0.054908 0.000584 0.000337 HXaCHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 58 + 46 −0.025062 0.000831 0.000480 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_Ami2638_CWT-LST 52 + 34 + 46 −0.023737 0.000656 0.000379 HXaCHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 58 + 46 −0.018786 0.000215 0.000124 HXaCHAPK_CHAPK_CWT-LST HXaM23-LST_CWT-LST HXaAmi2638_CWT-LST 28 + 34 + 70 −0.051336 0.000409 0.000236 HXaCHAPK_CWT-LST HXaM23-LST_M23-LST_CWT-LST