Surface display of functional proteins in a broad range of gram negative bacteria
10683509 ยท 2020-06-16
Assignee
Inventors
- Joachim Jose (Duesseldorf, DE)
- Mark George Teese (Muenster, DE)
- Shanna Sichwart (Muenster, DE)
- Iasson Tozakidis (Muenster, DE)
Cpc classification
C07K2319/60
CHEMISTRY; METALLURGY
C12Y302/01004
CHEMISTRY; METALLURGY
C12Y302/01091
CHEMISTRY; METALLURGY
C07K2319/035
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
C12N15/625
CHEMISTRY; METALLURGY
C12N9/2437
CHEMISTRY; METALLURGY
International classification
C12N15/10
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a method for the surface display of a recombinant polypeptide on the surface of a host cell, said method comprising the steps: (a) providing a host cell transformed with a nucleic acid fusion operatively linked with an expression control sequence, said nucleic acid fusion comprising: (i) a portion encoding a signal peptide, (ii) a portion encoding the recombinant polypeptide to be displayed, (iii) a portion encoding a transmembrane linker, and (iv) a portion encoding the trans porter domain of an EhaA protein, and (b) culturing the host cell under conditions wherein the nucleic acid fusion is expressed and the expression product comprising the recombinant polypeptide is displayed on the surface of the host cell.
Claims
1. A method for displaying a recombinant polypeptide on the surface of a host cell, said method comprising the steps: (a) providing a host cell transformed with a nucleic acid fusion operatively linked with an expression control sequence, said nucleic acid fusion comprising: (i) a portion encoding a signal peptide allowing for transport into the periplasm through the inner cell membrane, (ii) a portion encoding the recombinant polypeptide to be displayed, (iii) a portion encoding a transmembrane linker, and (iv) a portion encoding the transporter domain of an EhaA protein, and (b) culturing the host cell under conditions wherein the nucleic acid fusion is expressed and the expression product comprising the recombinant polypeptide is displayed on the surface of the host cell, wherein the host cell is a Gram negative bacterium, with the proviso that the Gram negative bacterium is not E coli, wherein the transporter domain of the EhaA protein is encoded by a sequence comprising a sequence selected from the group consisting of: (1) a nucleotide sequence comprising SEQ ID NO:18, (2) a nucleotide sequence encoding SEQ ID NO:19, (3) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:18 or/and a sequence encoding SEQ ID NO: 19, and (4) nucleotide sequences which encode the polypeptides encoded by (1), (2) or/and (3) within the scope of the degeneracy of the genetic code; wherein the transmembrane linker is encoded by a sequence comprising a sequence selected from the group consisting of (I) a nucleotide sequence comprising SEQ ID NO:16, (II) a nucleotide sequence encoding SEQ ID NO:17, (III) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:16 or/and a sequence encoding SEQ ID NO: 17, and (IV) nucleotide sequences which encodes the polypeptides encoded by (I), (II) or/and (III) within the scope of the degeneracy of the genetic code.
2. The method of claim 1, wherein the nucleic acid fusion further comprises at least one nucleic acid sequence encoding an affinity tag.
3. The method of claim 2, wherein a nucleic acid sequence defined in claim 2 is flanking the portion (ii) encoding the recombinant polypeptide to be displayed.
4. The method of claim 3, wherein the nucleic acid sequence is separated from portion (ii) by a sequence encoding at least one protease recognition sequence.
5. The method of claim 4, wherein the at least one protease recognition sequence is independently selected from factor Xa cleavage site, OmpT cleavage site, and TEV protease cleavage site.
6. The method of claim 2, wherein the affinity tag is independently selected from His.sub.6 and epitopes.
7. The method of claim 1, wherein the nucleic acid fusion comprises a nucleotide sequence encoding at least one protease recognition sequence, said nucleotide sequence being located between portions (ii) and portion (iii).
8. The method of claim 7, wherein the at least one protease recognition sequence is independently selected from factor Xa cleavage site, OmpT cleavage site, and TEV protease cleavage site.
9. The method of claim 1, wherein the portion (ii) encoding the recombinant polypeptide to be displayed is flanked by at least one sequence comprising a multiple cloning site.
10. The method according to claim 1 wherein the transporter domain of the EhaA protein forms a -barrel structure.
11. The method of claim 1, wherein the transmembrane linker (ii) comprises a sequence selected from the group consisting of: (a) an amino acid sequence comprising SEQ ID NO:17, and (b) sequences which are at least 95% identical to the sequences of (a).
12. The method of claim 1, wherein the transporter domain of the EhaA protein (iii) comprises a sequence selected from the group consisting of: (a) an amino acid sequence comprising SEQ ID NO:19, and (b) sequences which are at least 95% identical to the sequence of (a).
13. The method of claim 1, wherein the sequence of the nucleic acid fusion has a codon usage adapted to the host cell.
14. The method of claim 1, wherein the amino acid sequences encoded by nucleic acid sequences (i) to (iv) are arranged from N terminal to C terminal.
15. The method of claim 1, wherein the nucleic acid sequences (i) to (iv) are arranged from 5 to 3.
16. The method of claim 1, wherein the transporter domain of an EhaA protein is heterologous with respect to the host cell.
17. The method of claim 1, wherein the host cell is a bacterium.
18. The method of claim 1, wherein the bacterium is a Gram negative bacterium.
19. The method of claim 1, wherein the bacterium is selected from Salmonella spp., Zymomonas spp., Zymobacter spp., Pseudomonas spp., Cupriavidus spp., Rhodobacter spp., Acinetobacter spp., Gluconobacter spp., Gluconacetobacter spp., Acidomonas spp., Acetobacter spp., Paracoccous spp., Rhizobium spp., and Xanthomonas spp.
20. A recombinant vector comprising the nucleic acid fusion as defined in claim 1, operatively linked to an expression control sequence.
21. A recombinant vector comprising: (i) a portion encoding a signal peptide allowing for transport into the periplasm through the inner cell membrane, (ii) a portion encoding a multiple cloning site, (iii) a portion encoding a transmembrane linker, and (iv) a portion encoding the transporter domain of an EhaA protein, wherein the transporter domain of the EhaA protein is encoded by a sequence comprising a sequence selected from the group consisting of: (1) a nucleotide sequence comprising SEQ ID NO:18, (2) a nucleotide sequence encoding SEQ ID NO:19, (3) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:18 or/and a sequence encoding SEQ ID NO: 19, and (4) nucleotide sequences which encode the polypeptides encoded by (1), (2) or/and (3) within the scope of the degeneracy of the genetic code, and wherein the transmembrane linker is encoded by a sequence comprising a sequence selected from the group consisting of: (I) a nucleotide sequence comprising SEQ ID NO:16, (II) a nucleotide sequence encoding SEQ ID NO:17, (III) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:16 or/and a sequence encoding SEQ ID NO: 17, and (IV) nucleotide sequences which encodes the polypeptides encoded by (I), (II) or/and (III) within the scope of the degeneracy of the genetic code.
22. The vector of claim 21, wherein the multiple cloning site is suitable for integration of a nucleic acid sequence encoding a recombinant polypeptide in frame with portions (i), (ii) and (iv).
23. The vector of claim 21, wherein the nucleic acid sequences (i) to (iv) are arranged from 5 to 3.
24. A recombinant host cell comprising the recombinant vector as claimed in claim 21.
25. A method for producing a host cell capable of displaying a recombinant polypeptide on the surface, said method comprising the steps (a) providing a vector comprising: (i) a portion encoding a signal peptide allowing for transport into the periplasm through the inner cell membrane, (ii) a portion encoding a multiple cloning site, (iii) a portion encoding a transmembrane linker, and (iv) a portion encoding the transporter domain of an EhaA protein (b) inserting a sequence encoding the recombinant polypeptide to be displayed into the multiple cloning site (iv), and (c) performing the method of claim 1, wherein the transporter domain of the EhaA protein is encoded by a sequence comprising a sequence selected from the group consisting of: (1) a nucleotide sequence comprising SEQ ID NO:18, (2) a nucleotide sequence encoding SEQ ID NO:19, (3) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:18 or/and a sequence encoding SEQ ID NO: 19, and (4) nucleotide sequences which encode the polypeptides encoded by (1), (2) or/and (3) within the scope of the degeneracy of the genetic code, and wherein the transmembrane linker is encoded by a sequence comprising a sequence selected from the group consisting of: (I) a nucleotide sequence comprising SEQ ID NO:16, (II) a nucleotide sequence encoding SEQ ID NO:17, (Ill) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:16 or/and a sequence encoding SEQ ID NO: 17, and (IV) nucleotide sequences which encodes the polypeptides encoded by (I), (II) or/and (III) within the scope of the degeneracy of the genetic code.
26. A method for displaying a recombinant polypeptide on the surface of a host cell, said method comprising the steps: (a) providing a host cell transformed with a nucleic acid fusion operatively linked with an expression control sequence, said nucleic acid fusion comprising: (i) a portion encoding a signal peptide allowing for transport into the periplasm through the inner cell membrane, (ii) a portion encoding the recombinant polypeptide to be displayed, (iii) a portion encoding a transmembrane linker, and (iv) a portion encoding the transporter domain of an EhaA protein, and (b) culturing the host cell under conditions wherein the nucleic acid fusion is expressed and the expression product comprising the recombinant polypeptide is displayed on the surface of the host cell, wherein the recombinant polypeptide to be displayed is selected from an endoglucanase, an exoglucanase and a -glucosidase, and combinations thereof, wherein the host cell is a Gram negative bacterium, and wherein the transporter domain of the EhaA protein is encoded by a sequence comprising a sequence selected from the group consisting of: (1) a nucleotide sequence comprising SEQ ID NO:18, (2) a nucleotide sequence encoding SEQ ID NO:19, (3) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:18 or/and a sequence encoding SEQ ID NO: 19, and (4) nucleotide sequences which encode the polypeptides encoded by (1), (2) or/and (3) within the scope of the degeneracy of the genetic code, and wherein the transmembrane linker is encoded by a sequence comprising a sequence selected from the group consisting of: (I) a nucleotide sequence comprising SEQ ID NO:16, (II) a nucleotide sequence encoding SEQ ID NO:17, (III) nucleotide sequences comprising a sequence being at least 95% identical to SEQ ID NO:16 or/and a sequence encoding SEQ ID NO: 17, and (IV) nucleotide sequences which encodes the polypeptides encoded by (I), (II) or/and (III) within the scope of the degeneracy of the genetic code.
27. A method for displaying a recombinant polypeptide on the surface of a host cell according to claim 1, wherein the sequence of the nucleic acid fusion has a codon usage adapted to the host cell.
28. A method for displaying a recombinant polypeptide on the surface of a host cellusing a Maximized Autotransporter Expression System, said method comprising: (a) providing a host cell transformed with a nucleic acid fusion operatively linked with an expression control sequence, said nucleic acid fusion comprising: (i) a portion encoding a signal peptide which is a cholera toxin B subunit signal peptide, (ii) a portion encoding the recombinant polypeptide to be displayed, wherein said recombinant polypeptide is selected from the group consisting of an endoglucanase, an exoglucanase and a -glucosidase, (iii) a portion encoding a transmembrane linker, wherein said transmembrane linker is encoded by a sequence comprising a sequence selected from the group consisting of: (1) a nucleotide sequence comprising SEQ ID NO:16, (2) a nucleotide sequence encoding SEQ ID NO:17, (3) a nucleotide sequence comprising a sequence which is at least 95% identical to SEQ ID NO:16 and/or a sequence encoding SEQ ID NO: 17, and (4) nucleotide sequences which encode the polypeptides encoded by (1) or/and (2) within the scope of the degeneracy of the genetic code, (iv) a portion encoding the transporter domain of an EhaA protein, wherein the transporter domain of the EhaA protein is encoded by a sequence comprising a sequence selected from the group consisting of: (A) a nucleotide sequence comprising SEQ ID NO:18, (B) a nucleotide sequence encoding SEQ ID NO:19, (C) a nucleotide sequence comprising a sequence which is at least 95% identical to SEQ ID NO:18 and/or a sequence encoding SEQ ID NO: 19, and (D) a nucleotide sequence which encodes the polypeptides encoded by (A), (B) or/and (C) within the scope of the degeneracy of the genetic code, and (b) culturing the host cell under conditions wherein the nucleic acid fusion is expressed and the expression product comprising the recombinant polypeptide is displayed on the surface of the host cell, wherein the host cell is a Gram negative bacterium, with the proviso that the Gram negative bacterium is not E coli.
29. The method according to claim 1, wherein said recombinant polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, and SEQ ID NO:25.
30. The method of claim 1, wherein the signal peptide is a CtxB signal peptide.
31. The method of claim 1, wherein the signal peptide is obtained from a gram-negative bacterium.
Description
(1) Further, the present invention shall be further illustrated by the following figures and examples:
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(16) KT2440 strains. Cells were grown until OD.sub.600 0.6, and protein expression induced by 0.5% (w/v) Larabinose for 2 h at 30 C. (200 rpm). Cells were either used directly for preparations of outer membranes or with a prior incubation step with proteinase K and subsequent outer membrane preparation. (A)
(17) Coomassie stain and (B) esterase activity stain after renaturation of enzymes. M: PageRuler prestained protein marker, 1: control, E. coli UT5600 without plasmid, 2: control, proteinase K treated E. coli UT5600 without plasmid, 3: E. coli UT5600 pMATE-SI010 (EstA as passenger), 4: proteinase K treated E. coli UT5600 pMATE-SI010, 5: P. putida KT2440 pMATE-SI010, 6: proteinase K treated P. putida KT2440, 7: control, P. putida KT2440 pMATE-SI005 (6His as passenger), 8: control, proteinase KK treated P. putida KT2440 pMATE-SI005.
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(36) KT2440 pMATE--glucosidase with L-arabinose. The arrow indicates the band associated with the pMATE fusion protein.
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(43) TABLE-US-00019 Feature position AraC 4-902 bp araBAD promoter 912-1184 bp CtxB SP sequence 1227-1307 bp 6xHis 1308-1325 bp fXa cleavage site 1326-1337 bp exoglucanase 1338-3719 bp OmpT cleavage site 3720-3731 bp fXa cleavage site (2.nd) 3732-3743 bp PEYFK epitope 3747-3761 bp MATE linker and -barrel 3762-5228 bp pBBR1 rep gene (broad host rep) 5550-6212 bp KanR (kanamycine resistence cassette) 7746-8540 bp
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(45) TABLE-US-00020 Feature position AraC 4-902 bp araBAD promoter 912-1184 bp CtxB SP sequence 1227-1307 bp 6xHis 1308-1325 bp fXa cleavage site 1326-1337 bp endoglucanase 1338-2759 bp OmpT cleavage site 2760-2771 bp fXa cleavage site (2.nd) 2772-2783 bp PEYFK epitope 2787-2801 bp MATE linker and -barrel 2802-4268 bp pBBR1 rep gene (broad host rep) 4590-5252 bp KanR (kanamycine resistence cassette) 6786-7580 bp
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(47) TABLE-US-00021 Feature position AraC 4-902 bp araBAD promoter 912-1184 bp CtxB SP sequence 1227-1307 bp 6xHis 1308-1325 bp fXa cleavage site 1326-1337 bp -glucosidase 1338-2690 bp OmpT cleavage site 2691-2702 bp fXa cleavage site (2.nd) 2703-2714 bp PEYFK epitope 2718-2732 bp MATE linker and -barrel 2733-4199 bp pBBR1 rep gene (broad host rep) 4521-5183 bp KanR (kanamycine resistence cassette) 6717-7511 bp
EXAMPLE 1
(48) Surface Expression of 6His in E. coli using the MATE System
(49) In this example, the MATE system is used to display a short peptide (6His) on the surface of E. coli.
(50) Bacterial Strains
(51) E. coli BL21 (8,
(52) Plasmid Construction
(53) The gene encoding the pMATE autotransporter fusion protein was synthesised commercially in the pJexpress-401 plasmid vector (DNA2.0, USA) to create pMATEMT004. The pJExpress401 plasmid backbone contained an rrnB1/B2 terminator, kanamycin resistance gene, LacI repressor, pUC on as well as rpn and bla terminators. Expression of the fusion protein was under the transcriptional control of an Isopropyl -D-1-thiogalactopyranoside (IPTG) inducible T5 promoter.
(54) The fusion protein included an N-terminal signal peptide from the cholera toxin B subunit (CtxB), a 6His tag, a multiple cloning site, and the autotransporter domain, which consists of a linker and Pbarrel region.
(55) Protease cleavage sites (Factor Xa and OmpT) were incorporated after the multiple cloning site. A second Factor Xa cleavage site was inserted after the 6His, for removal of this affinity tag after purification. An epitope (PEYFK) for a monoclonal antibody (D142) was inserted after the protease cleavage sites.
(56) The pMATE contains the EhaA autotransporter domain (GenBank Accession No. Q8X6C1). This autotransporter domain has never before been used for the display of recombinant proteins in bacteria; however the full native protein is known to increase cell-cell interactions when overexpressed in E. coli (Wells et al. 2008). To define the border between the original passenger and the autotransporter domain, we aligned the EhaA polypeptide sequence against the Cterminal AIDAI fragment used for Autodisplay (GenBank Accession No. Q03155, Maurer et al. 1997). The sequence encoding the signal peptide and EhaA fragment was codon optimised for E. coli according the algorithm of Welch et al. (2009). The codon usage was further altered to remove some restriction sites, and to minimise predicted RNA 2 structure in the region encoding the CtxB signal peptide.
(57) SDS-PAGE and Western Blot 40 ml of LB media was inoculated with 1 ml from an overnight culture of E. coli BL21 or BL21 (DE3) containing the plasmid of interest. The cultures were then incubated at 37 C., 200 rpm until they reached an OD.sub.600 of 0.5. Protein expression was induced by the addition of 1 mM IPTG, and the cells harvested one hour after induction. Outer membrane proteins were prepared according to the rapid isolation protocol of Hantke (1981) with modifications as described previously (Jose and von Schwichow, 2004). Proteins were separated with 10% sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). Proteins were visualised after SDS-PAGE by staining with Coomassie Brilliant Blue R250. For Western blots, proteins were transferred onto a nitrocellulose membrane using standard electroblotting techniques (Mini-trans blot, Bio-Rad, USA). For the 1 antibody we used a mouse monoclonal anti-6-His IgG (Dianova, Germany). For the 2 antibody we used a horseradish peroxidase-conjugated anti-mouse IgG (Antibodies-online, USA). All solutions for Western blotting were based on Tris/Cl buffered saline (TBS, pH 7.4). The membrane was first blocked with 3% BSA, and incubated with a 1:1000 of 1 antibody for 3 hours. The membrane was then washed in TBS, and incubated for 2 hours with a 1:6000 dilution of 2 antibody. The blot was then washed and incubated with Pierce enhanced chemiluminescent (ECL) western blotting substrate (Thermo Scientific, USA), exposed to X-ray film.
Results and Discussion
(58) SDS-PAGE and Western blots revealed a protein of 60-65 kDa in the outer membrane of E. coli containing pMATE-MT004 (
(59) Overall, this example shows that the MATE system can transfer a peptide to the outer membrane of E. coli. It shows that the 6His is a suitable epitope for Western blotting, in order to detect the location of the fusion protein after SDS-PAGE.
EXAMPLE 2
(60) Surface Expression of GFP in E. coli using the MATE System
(61) In this example, the MATE system is used to display a fulllength protein (GFP) on the surface of E. coli.
(62) Bacterial Strains
(63) E. coli Stellar cells were used for cloning experiments [F, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, 80d lacZ M15, (lacZYAargF) U169, (mrrhsdRMSmcrBC), mcrA, ] (Clontech, USA). Expression experiments were carried out in Escherichia coli UT5600 [F.sup., ara-14, leuB6, secA6, lacY1, proC14, tsx-67, (ompT-fepC)266, entA403, trpE38, rfbD1, rpsL109(Str.sup.r), xyl-5, mtl-1, thi-1] (Grodberg and Dunn, 1988).
(64) Plasmid Construction
(65) The gene encoding GFP (GFPmut2) was inserted into pMT004 to form pMATE-MT006 (
(66) Protease Accessibility Assay
(67) E. coli UT5600 cells containing pMATE-MT006 were grown in LB medium to OD.sub.600 of 0.5. Protein expression was induced by the addition of 1 mM IPTG, and the cells were harvested after 1 hour. For trypsin treatment, cells were incubated with 1.2 g.Math.ml.sup.1 of Trypsin (6000 NFU.ml.sup.1) while shaking for 1 hour at 37 C. Outer membrane proteins were isolated, and proteins were separated by 12.5% SDS-PAGE. Gels were stained for total protein with Coomassie Brilliant Blue R-250. For Western blots, proteins were transferred to PVDF membranes (Mini-trans blot, Bio-Rad, USA). The blot was blocked in TBS containing 5% milk powder, and incubated with a 1:2000 dilution of polyclonal rabbit anti-GFP IgG (GeneTex #GTX26556) overnight at 4 C. while shaking. The membrane was then washed with 0.1% Tween in TBS, and incubated for 2 hours with a 1:10 000 dilution of 2 antibody, horseradish peroxidase coupled anti-rabbit IgG (Promega #W401B). Horseradish peroxidase activity was detected with luminol reagent (sc-2048, SantaCruz Biotechnology, USA) and a chemiluminescence imager (Chemocam, Intas).
(68) Pierce enhanced chemiluminescent (ECL) western blotting substrate (Thermo Scientific, USA), and viewed with an ECL imager.
(69) Results and Discussion
(70) A protein of approximately 105 kDa was found in the outer membrane in cells expressing GFP in the MATE system in E. coli. The apparent molecular weight is higher than the expected size of the fusion protein after cleavage of the Nterminal signal peptide (84 kDa). The 105 kDa band was detected with the antiGFP antibody (
(71) We conducted a protease accessibility test to confirm the passenger domain was exposed to the surface. The protease trypsin is too large to enter the cell, therefore the trypsin accessibility of an Nterminal passenger domain demonstrates surface exposure. In contrast, Nterminal regions that are inaccessible to trypsin are presumed to be translocation intermediates, or misfolded proteins in the cytoplasm.
(72) The band corresponding to the MATE fusion protein was significantly reduced after trypsin treatment (
EXAMPLE 3
(73) Secretion of a Recombinant Protein into the Medium using the MATE System
(74) In this example, the MATE system utilises the OmpT protease to enable the secretion of a fulllength protein.
(75) In this example, we show that GFP is secreted into the growth media after when expressed in OmpT positive strains using the MATE system.
(76) Materials and Methods
(77) pMATE-MT006 was transformed into E. coli UT2300 [F.sup., ara-14, leuB6, secA6, lacY1, proC14, tsx-67, entA403, trpE38, rfbD1, rpsL109(Str), xyl-5, mtl-1, thi-1] and its OmpT deficient derivate E. coli UT5600 [F.sup., ara-14, leuB6, secA6, lacY1, proC14, tsx-67, (ompT-fepC)266, entA403, trpE38, rfbD1, rpsL109(Str.sup.r), xyl-5, mtl-1, thi-1] (Grodberg and Dunn, 1988). 40 ml of LB medium was inoculated with 0.4 ml of an overnight culture, and grown until OD.sub.600 0.5 at 37 C., 200 rpm. 1 mM of IPTG was added, and the cells were incubated for another 1.5 hours. Cells were then removed by centrifugation (3750 g, 30 min, 4 C.) followed by vacuum filtration (0.45 m HVLP membrane, Millipore).
(78) Proteins secreted into the LB media were concentrated by TCA/Acetone precipitation. TCA (80% w/v) was added to the 40 ml of LB medium to a final concentration of 8.5%. The sample was then incubated for 1 h at 4 C., followed by centrifugation (3750 g, 30 min, 4 C.). The supernatant was then discarded except for a small amount (3 ml) which was used to resuspend the pellet and transfer to 1.5 ml tubes. After centrifugation at 18 000 g for 30 min, the pellet was resuspended in 1 ml of icecold acetone and followed by centrifugation. The pellet was then resuspended in icecold acetone in water (80% v/v) followed by centrifugation. The pellet was then dried for 20 min on a 37 C. heating block, and resuspended in 200 l of 1SDS sample buffer containing 100 mM of dithiothreitol (DTT). Proteins were dissolved by heating at 96 C. for 50 min with vigorous vortexing.
(79) Proteins were separated by 12.5% SDS-PAGE and proteins visualised with Coomassie Brilliant Blue R250. Western blotting was conducted using a 1 antibody against GFP (rabbit polyclonal anti-GFP, #GTX26556, GeneTex) and a 2 goat anti-rabbit IgG antibody, coupled with horseradish peroxidase (#W401B, Promega). After SDS-PAGE, proteins were transferred onto a PVDF membrane using standard electroblotting techniques (Mini-trans blot, Bio-Rad). All solutions for Western blotting were based on Tris/Cl buffered saline (TBS, pH 7.4). The membrane was blocked for 1 h with 5% milk powder (blotting grade, Roth). It was then incubated for overnight in a 1:1000 dilution of 1 antibody, at 4 C. while shaking. The membrane was then washed with 0.1% Tween in TBS and incubated for 1.5 hours with a 1:6000 dilution of 2 antibody. Horseradish peroxidase activity was detected with luminol reagent (sc-2048, SantaCruz Biotechnology, USA) and a chemiluminescence imager (Chemocam, Intas).
(80) Results and Discussion
(81) To facilitate secretion via OmpT cleavage, we inserted an artificial OmpT cleavage site (Ala-Arg-Arg-Ala) into the MATE autotransporter in between the passenger and the linker. Because the EhaA autotransporter region has never before been used for surface display of a recombinant passenger, we were unsure whether OmpT would facilitate cleavage, and we were also unsure if the autotransporter domain would facilitate self-proteolysis as seen for autotransporters in the SPATE family.
(82) To test if this artificial OmpT cleavage site could allow protein secretion, we transferred the plasmid pMATE-MT006 into OmpT positive strain UT2300 and looked for the presence of GFP in the growth media. Western blotting clearly showed the presence of GFP in the growth media of E. coli UT2300 pMATE-MT006 (
(83) When testing a new autotransporter system, it is important to determine if the surface displayed passengers are naturally released into the media by self-proteolysis. Self-proteolysis would reduce the amount of protein at the surface, and therefore reduce efficiency in biocatalysis or screening. In the MATE system we found no evidence that the passengers are released into the growth media by self-proteolysis. After surface expression in an OmpT negative strain (E. coli UT5600), GFP could be detected clearly in the outer membrane (see
(84) Overall, our experiments show the feasibility of OmpT mediated secretion of recombinant passengers using the MATE system. OmpT did not cleave the passenger protein GFP, despite the presence of a typical OmpT recognition site within the primary sequence. We show for the first time that an artificial OmpT cleavage site can be used for the release of surface displayed passengers. In comparison to SPATE-like autoproteolytic cleavage, release by OmpT has the advantage that the same plasmid can be used for both surface display and secretion by simply varying the expression strain. This might have advantages in high-throughput approaches for protein expression and screening.
EXAMPLE 4
(85) Secretion and Purification of Functional mCherry Protein using the MATE System in E. coli
(86) Introduction
(87) In this example, we show that mCherry is secreted into the growth media after expression in OmpT positive strains using the MATE system. The outer membrane protein T (OmpT) of E. coli is a surface membrane serine protease and is the prototypical member of the omptin family of gram-negative bacteria (Mangel, Toledo et al. 1994). Recombinant passengers have previously been shown to be secreted into the media, after surface display using autotransporters in OmpT containing E. coli strains. In all previous cases, an OmpT cleavage site was found within the autotransporter linker region. In our case it was unknown whether OmpT would facilitate cleavage, as the autotransporter within the MATE system (EhaA) has not been previously tested for the recombinant expression of passenger proteins. For this reason, we inserted an artificial OmpT cleavage site (Ala-Arg-Arg-Ala) to the Cterminal region of the recombinant passenger. Sevastsyanovich et al. already described the secretion of red fluorescent protein (RFP) mCherry by utilization of a serine protease autotransporter of the Enterobacteriaceae (SPATEs, Sevastsyanovich et al. 2012). In comparison to the cleavage procedure of the passenger domain of SPATES, the passenger of the MATE system is released by the endogenous protease OmpT by recognition of the artificial OmpT cleavage site within the plasmid.
(88) Materials and Methods
(89) Construction of pMATE-SI015
(90) A ligation independent cloning was used for construction of pMATE-SI015
(91) (In-Fusion Eco-Dry Kit, Clontech). The nucleotide sequence of pMATE-SI015 is described by SEQ ID NO:13 (
(92) (ATGATGGTGATGGTGGTGGGTGATGTTCTG). The gene encoding mCherry was amplified using the primers SI021 (AATAGCACGACGAGCcttgtacagctcgtccatgccgccggtgg) and PQ024 (CACCATCACCATCATATGGTGAGCAAGGGCGAGGAGGATAACATG), which contained a 15 by overlap to the PCR product of the pMATE-MT004 plasmid backbone, as required for InFusion cloning. The two PCR Products were joined to form the plasmid pMATE-SI015 using standard In-Fusion techniques (Eco-Dry kit, Clontech) and transformed into E. coli Stellar chemically competent cells.
(93) Bacterial Strains and Purification of mCherry
(94) pMATE-SI015 was transformed into E. coli UT5600 and its OmpT positive parent strain E. coli UT2300 (Grodberg and Dunn, 1988).
(95) 800 ml of LB medium was inoculated with 8 ml of an overnight culture, and grown until OD.sub.600 0.5 at 37 C., 200 rpm. 1 mM of IPTG was added, and the cells were incubated for 24 hours. Cells were then removed by centrifugation (10,000 g, 20 min, 4 C.).
(96) Proteins secreted into the LB media were purified via the 6His epitope present in the secreted heterologous mCherry passenger. Therefore the obtained supernatant was loaded on a Ni-NTA column, which was before equilibrated with 10 column volumes of binding buffer containing 20 mM sodium phosphate, 0.5 M sodium chloride and 20 mM imidazole (pH 7.0). The column was washed once with 10 column volumes binding buffer and with 30 ml washing buffer, containing 20 mM sodium phosphate, 0.5 M sodium chloride and 100 mM imidazole (pH 7.0). Elution of the protein was carried out by addition of three times 2 ml Elution buffer containing 20 mM sodium phosphate, 0.5 M sodium chloride and 500 mM imidazole (pH 7.0). The eluted protein fractions were concentrated in microcon centrifugal filter devices with a cut-off size of 10 kDA (Merck Millipore). All steps were carried out at 4 C.
(97) The concentrated protein was resuspended in 40 l of 100 mM sodium phosphate buffer pH 7.0 plus 40 l of 2SDS sample buffer containing 200 mM of dithiothreitol (DTT). Protein samples were heated at 96 C. for 10 min.
(98) Equal amounts of protein samples were analysed by 10% SDS-PAGE and visualised by Coomassie stain.
(99) Results and Discussion
(100) The outer membrane protein T (OmpT) of E. coli is a surface membrane serine protease and is the prototypical member of the omptin family of gram-negative bacteria (Mangel, Toledo et al. 1994).
(101) As described for the secretion of GFP using the MATE system in example 3a), we also tested the artificial OmpT cleavage site for the secretion of monomeric red flourescent protein (RFP) mCherry. Therefore we transferred the plasmid pMATE-SI015 into OmpT positive strain E. coli UT2300 and looked for the presence of mCherry in the growth media.
(102) Supernatants from cultures of both E. coli UT2300 and E. coli UT5600 showed no visible difference. We then attempted to purify mCherry from the supernatant using a Ni-NTA column. After applying the supernatant to the Ni-NTA column and elution with 500 mM imidazole the sample of OmpT-positive strain E. coli UT2300 was indeed pink. In constrast, repeating this procedure with the supernatant from OmpT-negative cells resulted in an eluate which was clear. This suggests that mCherry was indeed displayed at the surface and then released by the OmpT protease in the OmpT (
(103) The putative mCherry visualised by SDS-PAGE and subsequent Coomassie staining was visible as a single band with an apparent molecular weight of 30 kDa. In contrast, after expression and purification of the 6His proteins from the OmpT negative derivate strain E. coli UT5600 we did not detect any RFP in the growth media, suggesting by the absence of a band at the same molecular weight as it is the case for the OmpT positive strain (
(104) The presence of a band with an apparent molecular weight of 100 kDa for both, the OmpT positive and OmpT negative strain protein samples, suggests the co-purification of the full autotransporter fusion protein either after cell lysis or by occurrence of vesicles containing the autotransporter fusion protein (
(105) The size of the band from the eluate from E. coli UT2300 detected by SDS-PAGE with subsequent Coomassie stain strongly suggests that OmpT indeed cleaved at the artificial Ala-Arg-Arg-Ala site within the fusion protein and released the putative mCherry passenger. Overall, our experiments show the feasibility of OmpT mediated secretion of recombinant passengers using the MATE system. As it is described in Example 3a) for the GFP passenger, we did not detect any undesired cleavage of the mCherry passenger by OmpT. This example gives a further proof that the artificial OmpT cleavage site can be used for the release of surface displayed passengers. The OmpT mediated release has the advantage that the same plasmid can be used for both surface display and secretion. This allows greater flexibility than self-proteolysis, where it would be necessary to construct a second plasmid for surface display in which the residues necessary for self-proteolysis are removed.
EXAMPLE 5
(106) Release of Recombinant Passenger using Factor Xa, after Surface Display using the MATE System
(107) In this example, the MATE system is shown to enable the release of a fulllength protein using the specific protease Factor Xa.
(108) We showed earlier that the MATE system could be used for the constitutive secretion of a recombinant passenger in OmpT positive strains (Example 3). The secretome of E. coli is known to be relatively simple, reducing the costs associated with purification in comparison to intracellularly expressed proteins. However protein purification is still required to separate the recombinant protein of interest from other secreted proteins. For high-throughput screening, it would be beneficial to obtain small amounts of the protein of interest at higher purity. This could be achieved by first displaying the protein of interest on the cell surface, washing the cells in buffer, and then releasing the protein of interest into the buffer by the activity of a specific protease. The protein of interest can then be immediately assayed for functional activity. Optimally, the protease used to release the passenger is highly specific. In this example, we show that the protease Factor Xa can successfully release passengers displayed on the cell surface with the MATE system.
(109) E. coli UT5600 cells containing pMATE-MT006 were grown in an overnight culture in LB medium. 20 ml of LB medium was inoculated with 0.5 ml of overnight culture, and grown at 37 C., 200 rpm to OD.sub.600 of 0.5. Protein expression was induced by the addition of 1 mM IPTG, and the cells were harvested after 1.5 hour. Cells were washed in buffer consisting of 100 mM Tris/Cl pH 8, 50 mM NaCl, 1 mM CaCl.sub.2, and incubated for 16 h with 100 g.Math.ml.sup.1 of bovine Factor Xa protease (#33233, QIAGEN). Cells were removed by centrifugation at 14 000 g, and proteins within the supernatant were concentrated by TCA precipitation (final TCA concentration of 8%). Concentrated proteins from the supernatant were separated by 12.5% SDS-PAGE. Western blots were performed with polyclonal rabbit anti-GFP and horseradish peroxidase-coupled anti-rabbit IgG as described in Example 3.
(110) Soluble GFP (28 kDa) was detected in the supernatant after Factor Xa treatment of E. coli pMATE-MT006 (
(111) In combination with the 6His on the Nterminal region of the passenger, the selective release in the MATE system allows the rapid purification of proteins. As shown here, the cells can be washed before the release of the passenger into a buffer. This yields a purer form of the protein in comparison to a constitutive secretion system such as auto-proteolysis, where the passenger is released into the growth media and must be separated from other secreted proteins.
(112) The Factor Xa mediated release also allows a more rapid protease-accessibility assay in the MATE system. Usually a nonspecific protease such as trypsin or proteinase K is used, whereby the incubation time must be optimised for each system. Proteins transported to the surface with Autodisplay have also been released with purified Iga1 protease (Klauser 1992), however this enzyme is not commercially available and the mode of action is not extensively researched. Only recently has this been attempted with a commercially available, highly specific protease (Nla-TEV, Ko et al. 2012). Here, we show the detection of the passenger in the supernatant after incubation of the cells with Factor Xa, a rapid and simple method of proving surface display, without the need for antibodies or extensive optimisation of protease digestion. When optimised, it may be also possible to detect the decrease in cell-associated passenger after Factor Xa treatment. This technique allows rapid and simple protein purification, and may be compatible with highthroughput screening approaches for protein improvement or inhibitor detection.
EXAMPLE 6
(113) Functional Display of B. gladioli EstA Esterase on the Surface of E. coli with the MATE System
(114) In this example the EstA catalytic domain was functionally expressed on the surface of E. coli using the new MATE system.
(115) Plasmid Construction
(116) The plasmid pMATE-SI005 (
(117) The fusion protein encoded by pMATE-SI005 (SEQ ID NO:8) included a CtxB signal peptide, a small peptide passenger (6His), a multiple cloning site, OmpT and Factor Xa cleavage sites and a PEYFK epitope. The Cterminus contains the EhaA autotransporter linker and n-barrel.
(118) For the surface display of a functional esterase, the coding region of the catalytic domain of estA from Burkholderia gladioli was excised from pES01 (Schultheiss et al. 2002) via Xhol and Kpnl and subsequently inserted in pMATE-SI005 to create pMATE-SI010 (
(119) We conducted activity tests to determine if EstA catalytic domain was expressed in a functional form. For this purpose cells of E. coli UT5600 pMATE-SI010 and E. coli BL21(DE3) pES01 were cultivated at 30 C. E. coli UT5600 without any plasmid served as a control. E. coli UT5600 cells were induced with 0.5% (w/v) L-arabinose for 2 hours. Cells of E. coli BL21 (DE3) pES01 were also cultivated for 2 hours. Since pES01 plasmid encodes a promoter facilitating a constitutive gene expression there was no need for any induction. Photometric activity assays were performed with pnitrophenyl acetate as a model esterase substrate. To obtain standard conditions OD.sub.600 was adjusted to 0.2 for every measurement. In comparison to pES01 the MATE system gives vastly improved activity of surface-displayed esterase (
EXAMPLE 7
(120) Functional Surface Display of EstA Catalytic Domain in Salmonella enterica using the MATE System
(121) In this example the MATE system was used for expression of the estA catalytic domain in a second non E. coli strain, namely S. enterica serovar Typhimurium. Autotransporter-mediated surface display has been previously conducted in Salmonella strains (van Gerven et al. 2009), which are genetically closely related to E. coli. However this provides the first confirmation that the vector is compatible in non E. coli strains.
(122) The broad host range plasmid pMATE-SI010 (
(123) Proteinase K is too large to enter the cell envelope of S. enterica. This means, if the EstA catalytic domain is degraded by proteinase K, it must be accessible at the cell surface.
(124) Activity assays with pnitrophenyl acetate as a substrate were performed with either proteinase K treated or untreated whole cells containing S. enterica pMATE-SI010. Protein expression was induced for 2 h with 0.5% L-arabinose at 30 C. The final OD.sub.600 in the assay was adjusted to 0.2. We used S. enterica, without any plasmid as a negative control. Cells of S. enterica pMATE-SI010 exhibited similar activity in comparison to E. coli cells harbouring pMATE-SI010 plasmid (
(125) The decrease in esterase activity at the surface after proteinase K treatment confirms that the estA catalytic domain was exposed to the surface. This confirmed that the MATE system functions not only in E. coli, but also in a closely related non E. coli species. The use of the MATE system in S. enterica may help in the development of vaccines. To our knowledge, this is the first time that surface display of a recombinant protein with an autotransporter has been successfully achieved in a Salmonella species.
EXAMPLE 8
(126) Functional Surface Display of EstA Catalytic Domain in Pseudomonas putida KT2440 with the MATE System
(127) In this example the MATE system was used for the functional expression of and enzyme in a Gram negative species more distantly related to E. coli.
(128) Functional surface display of estA in E. coli was carried out as described in Example 4. The broad host range plasmid pMATE-SI010, encoding the catalytic domain of the esterase EstA, was transferred to cells of P. putida KT2440 using standard chemical transformation techniques.
(129) Functional activity was first tested using continuous esterase microplate assays. Esterase activity in P. putida cells containing pMATE-SI010 was significantly higher than cells containing the control plasmid, pMATE-SI005 (
(130) To confirm that this functional activity was at the cell surface, the esterase microplate assays were conducted with whole cells after incubation with proteinase K. Proteinase K is too large to cross the outer membrane. The loss of activity in whole cells after addition of proteinase K givesstrong evidence that the esterase is located at the cell surface. Using pnitrophenyl acetate as a substrate, activity assays were performed with either proteinase K treated or untreated whole cells of P. putida pMATE-SI010.
(131) P. putida cells expressing an esterase at the surface using the MATE system completely lost their esterase activity after treatment with proteinase K (
(132) We conducted flow cytometry analysis to confirm that the fusion protein containing the EstA fragment was exposed at the surface. The results are described in
(133) The fully processed, surface-displayed EstA fusion protein was predicted to have an N-terminus consisting of a 6His affinity tag, the EstA fragment and MATE autotransporter domain. Using an antibody against the 6His epitope, approximately 50% of the cells were found in a population with high fluorescence and could be judged to contain the fusion protein at the surface. To determine whether non-stained cells represented incomplete surface expression, or simply sub-optimal antibody binding, we conducted further analyses of cellular and outer membrane proteins.
(134) As with many other esterases, EstA can be refolded after SDS-PAGE and functional activity measured using an ingel activity stain (Schultheiss et al. 2002). Briefly, proteins were heated for 30 min in loading buffer that contained DTT, and separated by 10% SDS-PAGE as per standard techniques (Laemmli 1970). The gels were then incubated in buffer containing 2.5% Triton-X100 until the esterase had refolded into an active form. The gels were then stained for esterase activity using 1naphthyl acetate as a substrate and Fast Blue RR as a conjugate dye.
(135) In the analysis of whole cells expressing EstA at the surface, SDS-PAGE followed by esterase staining revealed a band at approximately 105 kDa in both E. coli and P. putida (
(136) A further SDS-PAGE analysis was conducted to confirm the surface exposure of the EstA esterase in P. putida, when expressed using the MATE system. To find out whether the EstA catalytic domain of the fusion protein was indeed exposed at the cell surface, proteinase K was added the whole cells containing pMATE-SI010 after protein induction with 0.5% (w/v) L-arabinose for two hours at 30 C. Proteinase K is too large of a molecule to enter the cell envelope of E. coli, the decrease in bands associated with the fusion protein after Coomassie or esterase staining indicates the surface exposure of the esterase. SDS-PAGE with Coomassie stain allows a further control: OmpA has a Nterminal extension in the periplasm that is proteinase K sensitive, therefore the detection of intact OmpA indicates that the protease has not entered the periplasm due to cell leakiness.
(137) After isolation of membrane proteins from P. putida, the EstAautotransporter fusion protein was visible after SDS-PAGE with both Coomassie and esterase staining (
(138) Confirming the results from microplate assays (
(139) In comparison to E. coli, the vast majority of esterase activity in P. putida pMATE-SI010 was protease accessible. This could suggest that P. putida has unexpected advantages for use with surface display. Another surprising benefit within P. putida was the absence of of the undesired intracellular fragments, which may indicate a small amount of the fusion protein is improperly folded in E. coli. These factors suggest that P. putida is highly suitable to screening of surface displayed enzymes or fluorescent molecules where intracellular activity is detrimental.
(140) When we directly compared the surface-exposed (i.e. protease accessible) esterase activity of E. coli and P. putida, the overall activity in E. coli was higher (
EXAMPLE 9
(141) Functional Surface Display of B. gladioli Esterase EstA Catalytic Domain in Zymomonas mobilis with the MATE System
(142) Introduction
(143) Zymomonas mobilis is of big interest for the commercial production of biomass-derived ethanol as it produces ethanol with high yields and has a higher growth rate than the presently used Sacharomyces cerevisiae. The exposure of recombinant proteins on the surface of Z. mobilis offers considerable advantages in the industrial application of this organism. For instance, the surface display of cellulose degrading enzymes (cellulases) on Z. mobilis cells can provide a whole-cell catalytic system which is able to breakdown cellulose and ferment the formed monomeric sugars to ethanol in a single step. As cellulose cannot penetrate the cell membrane, elaborate and expensive enzyme purification steps become necessary when the cellulases are expressed intracellularly. Surface exposed cellulases could circumvent these difficulties as they have direct access to their substrates and do not have to be extracted from the cells prior to their use. A further benefit of a whole-cell catalyst is given by its reusability, since bacterial cells can easily be separated from a reaction mixture by centrifugation.
(144) To our knowledge no surface display system for Z. mobilis has been established so far. This example presents the functional expression of B. gladioli esterase EstA catalytic domain on the surface of Z. mobilis using the new MATE system.
(145) Materials and Methods
(146) Construction of Plasmid pMATE-SI012 and Transfer to Z. mobilis
(147) For the construction of pMATE-SI012 (
(148) Cultivation of Z. mobilis and Induction of Protein Expression
(149) 200 mL of ZM Medium (10 g/L bacto peptone, 10 g/L yeast extract, 20 g/L glucose; Roth, Germany, when needed kanamycine 150 mg/mL; Sigma Aldrich, USA) were inoculated with an overnight-culture of Z. mobilis (1:1000 dilution) and incubated at 30 C., 60 rpm until the cultures reached an OD578 of 0.6. Induction of protein expression was performed by incubation with 0.2% (w/v) L-arabinose (Roth, Germany) in ZM Medium for two hours at 30 C., 60 rpm.
(150) Trypsin Digestion
(151) Z. mobilis cells were harvested by centrifugation and resuspended in 0.2 mol/L Tris-HCl pH 8.0. Porcine pancreatic trypsin was added to a final concentration of 2.5% and incubated for one hour at 37 C., 200 rpm. Digestion was stopped by washing three times with 10% fetal calf serum (FCS) in 0.2 mol/L Tris-HCl pH 8.0.
(152) Outer Membrane Protein Isolation
(153) Outer membrane protein isolation was carried out according to a modified protocol of Hantke (Hantke 1981, Jose and von Schwichow, 2004).
(154) Protein Separation
(155) Proteins were heated for 40 min at 95 C. in two-fold sample buffer (100 mM Tris-HCl, pH 6.8 containing 4% sodium dodecyl sulfate, 0.2% bromophenole blue, 20% glycerol and 30 mg dithiothreitol) and separated by means of sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a 10% resolving gel (Laemmli 1970). Proteins were then stained either with Coomassie Brilliant Blue or esterase activity stain (see below). Pictures were taken using an Intas Gel iX Imager (Intas, Germany).
(156) Western Blot
(157) Standard blotting techniques were used to transfer proteins from polyacrylamide-gels to nitrocellulose membranes (Mini-trans blot, Bio-Rad, USA). The membranes were blocked with 3% bovine serum albumine (BSA) in phosphate-buffered saline (PBS, pH 7.4) and incubated with mouse anti-6His mAb IgG1 (Dianova, Germany) in PBS (1:1000 dilution). Subsequently the membranes were washed three times with PBS and incubated with horseradish peroxidase-conjugated anti-mouse IgG (Antibodies-online, USA) in PBS (1:5000 dilution). The membranes were then washed twice with PBS and treated with Pierce enhanced chemiluminescent (ECL) western blotting substrate (Thermo Scientific, USA). Pictures were taken using an Intas ChemoCam Imager (Intas, Germany).
(158) Esterase Activity Staining
(159) Esterase activity staining was carried out using a modified protocol of Schultheiss et al. (Schultheiss 2002). For the renaturation of the esterase SDS was removed by incubation of the polyacrylamide gels in 10 mmol/L Tris-HCl, pH 7.5 containing 2.5% Triton-X100 (AppliChem, Germany) for three hours. The gels were then stained for esterase activity with 10 mmol/L
(160) Tris-HCl, pH 7.5 containing 0.1% (w/v) FastBlueRR (Sigma Aldrich, USA) and 2% (v/v) -naphtyl-acetate-solution (1% w/v in 50% v/v acetone; Sigma Aldrich, USA).
(161) Results
(162) SDS-PAGE Coomassie Staining
(163) Outer membrane proteins of Z. mobilis cells without plasmid pMATE-SI012, cells with plasmid and cells with plasmid and aforegoing trypsin treatment were isolated and separated by SDS-PAGE. Coomassie staining of proteins isolated from cells with plasmid did not show any additional bands assignable to the EstA-autotransporter fusion protein. This is attributed to the small proportion of fusion protein in relation to the total outer membrane protein amount. Coomassie staining of proteins isolated from cells that were treated with trypsin prior to the outer membrane protein isolation revealed the disappearing of most protein bands except of four bands, which are visible at apparent molecular weights of <25 kDa (a), 25-35 kDa (b) and 35-40 kDa (c and d) (see
(164) Western Blot
(165) Western Blot analysis of outer membrane protein isolates with anti-6His antibodies showed the presence of the EstA-autotransporter fusion protein at an apparent molecular weight of about 105 kDa, which is slightly higher than expected from the primary sequence (91.2 kDa). This is not unusual for beta-barrel containing proteins as they are not completely denatured under normal SDS-PAGE conditions. Neither in the outer membrane protein isolates of Z. mobilis cells without plasmid nor in the protein isolates of cells with plasmid but without L-arabinose induction a band was visible. Protein isolates of cells which were treated with trypsin prior to protein isolation also did not show any band. The absence of the fusion protein in the outer membrane proteins of trypsin-digested cells confirms the actual exposure of the esterase on the surface of the Z. mobilis cells (see
(166) Esterase Activity Stain
(167) In-gel esterase activity staining revealed an activity band at about 105 kDa in outer membrane proteins of arabinose treated Z. mobilis cells with plasmid. This is consistent with the presented Western Blot results, which identified this band as the EstA-autotransporter fusion protein. Besides this band, three other esterase activity bands between 55 and 100 kDa were visible in the same protein isolate. It is believed that these activity bands are unspecific degradation products. Esterase activity bands could neither be detected in protein isolates of cells without plasmid nor in protein isolates of cells with plasmid but without arabinose induction. Outer membrane proteins of cells that were trypsin digested also showed no esterase activity at all (see
(168) Conclusion
(169) Besides the successful establishment of the MATE system in P. putida and S. enterica, these experiments confirm the proper functionining of the MATE system in a third Zymomonas mobilis, a natural ethanol producer.
EXAMPLE 10
(170) Surface Display of an Active Endoglucanase Obtained from Bacillus subtilis on the Ethanologenic Bacteria Zymobacter palmae and Zymomonas mobilis using Maximized Autotransporter Expression (MATE) System
(171) Introduction
(172) The biocatalytic conversion of cellulose to ethanol as a doorway to alternative fuel production is to date limited by the absence of a catalytic system applicable to economic large scale processes. The biocatalyst of choice combines the ability to break down cellulose and simultaneously ferment the released sugars to ethanol. The high growth and ethanol production rates of the Gram-negative bacteria Zymobacter palmae and Zymomonas mobilis make them promising host organisms for such an approach. Since they cannot degrade cellulose naturally, it is necessary to add extracellular (i.e. secreted or cell-bound) endoglucanase, exoglucanase and beta-glucosidase functionalities to these organisms. In this example we describe the expression of a recombinant fusion protein consisting of an endoglucanase obtained from Bacillus subtilis and the maximized autotransporter expression (MATE) translocation unit in Z. palmae and Z. mobilis, resulting in the exposure of functional active endoglucanase on the cell surface of of both species.
(173) Construction of Plasmid pMATE-PT004
(174) The plasmid pMATE-PT004 was constructed based on the previously described pMATE-SI010 (SEQ ID NO:9). Plasmid pMATE-SI010 encodes a fusion protein consisting of an N-terminal CtxB signal peptide, Burkholderia gladioli esterase EstA catalytic domain and the C-terminal EhaA autotransporter domain. The expression of the fusion protein is controlled by an arabinose inducible promoter (araBAD). For detection and purification purposes, 6His and PEYFK-epitopes are incorporated N- and C-terminal of the esterase domain, respectively. The plasmid encodes a Kanamycin resistance gene for transformant selection (
(175) The esterase EstA catalytic domain of pMATE-SI010 was removed by restriction enzyme digestion with Xhol and Kpnl, while the endoglucanase domain was obtained by an identical restriction enzyme digestion of pMATE-endoglucanase and inserted into pMATE-SI010 to generate pMATE-PT004. Furthermore, a mob gene was inserted into pMATE-PT004 using ligation independent cloning methods (In-Fusion Cloning Kit HD, Clontech, USA) to enable plasmid replication in Z. mobilis and Z. palmae (
(176) Cultivation and Transformation of used Bacterial Strains
(177) Z. mobilis (DSM No. 3580) was cultured at 30 C., 80 rpm in culture medium containing 10 g/L bacto peptone, 10 g/L yeast extract and 20 g/L D-glucose. Z. palmae (DSM No. 10491) was cultured at 30 C., 200 rpm in growth medium containing 10 g/L yeast extract, 2 g/L potassium dihydrogen phosphate and 5 g/L sodium chloride, adjusted to pH=6.0. Insertion of plasmids was conducted according to standard electroporation procedure, electroporated cells were regenerated in culture medium for 6 h (Z. mobilis) and 2 h (Z. palmae), before transferring them to Kanamycin selection plates.
(178) Sodiumdodecylsulfate Polyacrylamide Gel Electrophoresis (SDS PAGE)
(179) Overnight cultures of Z. mobilis/Z. palmae containing pMATE-PT004 were used to inoculate 100 ml of the respective culture medium (1:100 dilution), and were incubated until the cultures reached OD.sub.578 of 0.5. Protein expression was induced by the addition of 0.2% L-arabinose, and the cells harvested one hour after induction. Outer membrane proteins were prepared according to the rapid isolation protocol of Hantke (Hantke 1981) with modifications as described previously (Jose and von Schwichow, 2004).
(180) Proteins were separated with 12.5% sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). Proteins were stained with Coomassie Brilliant Blue R250.
(181) Fluorescence-Activated Cell Sorting (FACS)
(182) Cells were cultivated as described above and adjusted to an OD.sub.578 of 0.4. After washing three times with cold, particle-free PBS, cells were resuspended in PBS containing 1 antibody against 6His (THE HisTag, mouse, GenScript, USA) in a dilution of 1:500. After 30 min incubation at 4 C., cells were washed three times with PBS and incubated 30 min with 2 antibody (goat anti-mouse IgG (H+L), Dylight 633 conjugated, Thermo Scientific Pierce Antibodies, Germany) at room temperature in the dark. Cells were washed again three times and cell fluorescence measured by FACS Aria III flow cytometer (BD Biosciences, USA) using a red laser for excitation at 633 nm and a 660/20 bandpass filter for detection.
(183) Whole Cell CMC Hydrolysis Activity Assay
(184) For the whole cell endoglucanase activity assay cells were cultivated as described above and adjusted to an OD of 25. Cells were incubated 10 min with 1% Carboxymethylcellulose (CMC) in sodium-citrate buffer, pH 6 at temperatures between 30 C. and 80 C. After cell removal, released reducing sugars were determined photometrically (absorption at 540 nm) using a modified 3,5-dinitrosalicylic (DNS) assay protocol of King et al. (2008). The absorption values of blank samples (buffer and substrate) were subtracted from the measured absorption values of the cell samples.
(185) Expression of Fusion Protein
(186) To confirm the expression of the endoglucanase fusion protein, outer membrane proteins of Z. palmae were isolated and separated by SDS-PAGE (
(187) Whole Cell CMC Hydrolysis Activity Assay
(188) Z. palmae wildtype cells, Z. palmae cells with pMATE-esterase (negative controls) and cells with pMATE-PT004 were analysed in terms of their CMC hydrolysis activity. Induced Z. palmae cells containing the pMATE-PT004 plasmid show high CMC hydrolysis activity compared to negative controls.
(189) CMC hydrolysis activity could be observed within a temperature range of 30 C. to 80 C. Non-induced cells with pMATE-PT004 show slightly higher activity than the negative controls, explained by the known effect of low basal protein expression level when using an arabinose promoter (
(190) Whole cell CMC hydrolysis activity assay was also performed with Z. mobilis wildtype and Z. mobilis pMATE-PT004 cells. Z. mobilis pMATE-PT004 cells showed high activity in a temperature range of 30 C. to 80 C. compared to Z. mobilis wild type cells (
(191) Since CMC is a large polymer which is not able to pass the cell wall, neither by diffusion nor by active transport, the observed hydrolysis of CMC represents strong evidence for the localisation of the enzyme on the cell surface.
(192) FACS
(193) FACS-histogram of Z. palmae pMATE-PT004 cells expressing the endoglucanase fusion protein (
(194) Conclusion
(195) We could successfully apply the MATE system to express a surface-displayed, active endoglucanase in the ethanologenic bacteria Zymomonas mobilis and Zymobacter palmae. This example illustrates the broad applicability of our invention to bacterial strains with prospective industrial use, in particular in combination with cellulose-degrading enzymes and ethanol-producing bacteria, forming a whole-cell biocatalyst for the production of second generation biofuels.
EXAMPLE 11
(196) Functional Display of Bacterial Cellulases on the Surface of Pseudomonas putida with the pMATE System
(197) In this example an endoglucanase obtained from Bacillus subtilis, an exoglucanase obtained from Clostridium thermocellum and a 3-glucosidase obtained from Clostridium thermocellum were functionally expressed on the surface of P. putida using the new pMATE system. Plasmids used for transformation of P. putida are described in
(198) SEQ ID NO:20 describes the nucleotide sequence of the autotransporter fusion gene encoded by pMATE-exoglucanase, for the surface display of an exoglucanase using the pMATE system.
(199) SEQ ID NO:21 describes the polypeptide sequence of the autotransporter fusion protein encoded by pMATE-exoglucanase, for the surface display of an exoglucanase using the pMATE system.
(200) SEQ ID NO:22 describes the nucleotide sequence of the autotransporter fusion gene encoded by pMATE-endoglucanase, for the surface display of an endoglucanase using the pMATE system.
(201) SEQ ID NO:23 describes the polypeptide sequence of the autotransporter fusion protein encoded by pMATE-endoglucanase, for the surface display of an endoglucanase using the pMATE system.
(202) SEQ ID NO:24 describes the nucleotide sequence of the autotransporter fusion gene encoded by pMATE--glucosidase, for the surface display of a -glucosidase using the pMATE system.
(203) SEQ ID NO:25 describes the polypeptide sequence of the autotransporter fusion protein encoded by pMATE--glucosidase, for the surface display of a -glucosidase using the pMATE system.
(204) The expression was tested by isolation of the outer membrane proteins and following separation by 10% SDS-PAGE (
(205) For activity assays cells were cultivated as described above. The exoglucanase activity was measured at pH 6 and 55 C. using 5 mM p-nitrophenyl-3-D-cellobioside as substrate. OD.sub.578 were adjusted to 0.5. After different incubation times cells were removed by centrifugation and the liberated p-nitrophenol in the supernatant was detected colorimetrically at 400 nm in order to determine the hydrolysis rate. In comparison to control cells, P. putida cells, expressing the exoglucanase, show continuous p-nitrophenol release within 4 min (
(206) The endoglucanase activity was measured by determining reducing sugars released of enzyme reaction with 1% carboxymethylcellulose (CMC) at pH 6 and 55. OD.sub.578 were adjusted to 20. After cell removal reducing sugars were detected in the supernatant colorimetrically at 540 nm using the 3,5-dinitrosalicylic acid (DNS) assay, modified by King et al (2008). In comparison to control cells, endoglucanase expressing P. putida cells show CMC-hydrolysis (
(207) The -glucosidase activity was measured at pH 6 and 55 C. using 5 mM p-nitrophenyl--D-glucopyranoside as substrate. OD.sub.578 were adjusted to 20.
(208) After different incubation times cells were removed by centrifugation and the liberated p-nitrophenol in the supernatant was detected colorimetrically at 400 nm in order to determine the hydrolysis rate. In comparison to control cells, P. putida cells, expressing the -glucosidase, show p-nitrophenol within 10 min (
(209) These examples show that the pMATE system can be used for functional display of cellulases on the surface of P. putida.
(210) The most common total cellulase activity assay is the filter paper assay
(211) (FPA) using Whatman No 1 filter paper as the substrate, which was established and published by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose 1987). To measure total pMATE-cellulase activity, exoglucanase, endoglucanase and -glucosidase were mixed in equal parts. OD.sub.578 of total cells were adjusted to 50. The FPA was performed at pH 6 and 55 C., modified by Xiao et al (2004). Reducing sugars, released by degradation of filter paper were detected in the supernatant colorimetrically at 540 nm using the 3,5-dinitrosalicylic acid (DNS) assay, modified by King et al (2008). In comparison to control cells, the mix of cellulase expressing P. putida cells show filter paper hydrolysis. Using glucose as standard the amount of released sugar equivalents (RSE) from filter paper after 4 days is 0.2 mg/ml (
(212) To determine the hydrolysis of a real lignocellulosic substrate by pMATE-cellulases, pretreated empty fruit punches (EFP) were used as substrate (2.5% dry weight). Exoglucanase, endoglucanase and -glucosidase expressing cells were mixed in equal parts. OD.sub.578 of total cells were adjusted to 20. The reaction was performed at pH 6 and 55 C. for 4 days. Reducing sugars (in particular glucose, cellubiose or/and cellulose-polysaccharide chains of variable length), released by EFB hydrolysis were detected in the supernatant colorimetrically at 540 nm using the 3,5-dinitrosalicylic acid (DNS) assay, modified by King et al (2008). In comparison to control cells, the mix of cellulase expressing P. putida cells show EFB degradation. Using glucose as standard the amount of released sugar equivalents (RSE) from from EFB after 4 days is 6.4 mg/ml (
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