Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficie...
Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V140, V176, K179, V226, V232, and K234, which were truncated from the C-terminal end of the mipA gene (MV140, MV176, MV179, MV226, MV232, and MV234) were examined. The whole-cell lipase activities showed that MV140 was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV140 as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV140. Additionally the MV140 motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV140 motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.
Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V140, V176, K179, V226, V232, and K234, which were truncated from the C-terminal end of the mipA gene (MV140, MV176, MV179, MV226, MV232, and MV234) were examined. The whole-cell lipase activities showed that MV140 was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV140 as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV140. Additionally the MV140 motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV140 motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
In this study, a C-terminal truncation strategy was used to display the protein of interest, and six cleavage sites (V140, C176, K179, V226, V232, and K234) of the mipA gene were tested as possible fusion sites from loops 3, 4 and 5 exposed on the exterior of the outer membrane.
2]. Comparison of lipase activity between the MipA anchoring motifs developed in this study and the previously reported motifs FadL [8], OprF [9], and YiaT [20]. E.
For construction of expression systems composed of truncated MipA fused to a target protein (Fig. 1B), the fulllength mipA gene, as well as the C-terminal truncated mipA (mipAt) genes encoding the first 140, 176, 179, 226, 232 and 234 amino acids from the N-terminus, were amplified by PCR using the primers shown in Table 2. The genes were cloned into the EcoRI and XbaI sites of pTrc99A to make pTrcM, pTrcMV 140 , pTrcMV 176 , pTrcMK 179 , pTrcMV 226 , pTrcMV 232 , and pTrcMK 234 , respectively.
In this study, we developed an efficient E. coli cell surface display using a novel anchoring motif truncated from the E. coli MltA-interacting protein (MipA; Swiss-Prot no. P0A908) at the C-terminus. To determine the best anchoring motif from MipA, several possible motifs were tested by creating truncated mipA genes to link the function of a protein, specifically a highly thermostable lipase from Pseudomonas fluorescens SIK W1 (49.
To display a lipase on the E. coli cell surface, the P. fluorescens lipase gene containing the FLAG sequence (DYKDDDDK) was amplified using primers 15 and 16; it was then cloned into the XbaI and HindIII sites of the pTrcMV 140 , pTrcMV 176 , pTrcMK 179 , pTrcMV 226 , pTrcMV 232 , and pTrcMK 234 vectors to create pTrcMV 140 PL, pTrcMV 176 PL, pTrcMK 179 PL, pTrcMV 226 PL, pTrcMV 232 PL, and pTrcMK 234 PL, respectively. E.
coli MipA protein in this work. To select the best anchoring site from MipA, six possible sites were tested by designing and constructing the lipase fusion display systems. Among these sites, the enzyme activities showed that MV140 was the best anchoring motif for the E.
대상 데이터
The proteins were transferred to Immobilon-P PVDF membranes (Millipore); the membranes were stained with MemCode reversible protein stain (Pierce Biotechnology) and imaged to verify that the protein loads were uniform and to ensure that efficient electrotransfer occurred, and the membranes were destained with Milli-Q water and blocked with nonfat dry milk prior to incubation with each primary antibody. For the immunodetection of the fusion protein, a monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich Co., USA) and a goat anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate (Sigma-Aldrich) were used. An enhanced chemiluminescence (ECL) kit (Amersham ECL Prime Western Blotting Detection Reagent; GE Healthcare Bio-Sciences AB, Sweden) was used for signal detection.
The cells were mounted on poly-L-lysine–coated microscopic slide glasses and examined by confocal microscopy (Carl Zeiss, Germany). Photographs were taken with a Carl Zeiss LSM 410. The samples were excited at 488 nm, and the images were filtered by a longpass 505-nm filter.
1B), the fulllength mipA gene, as well as the C-terminal truncated mipA (mipAt) genes encoding the first 140, 176, 179, 226, 232 and 234 amino acids from the N-terminus, were amplified by PCR using the primers shown in Table 2. The genes were cloned into the EcoRI and XbaI sites of pTrc99A to make pTrcM, pTrcMV 140 , pTrcMV 176 , pTrcMK 179 , pTrcMV 226 , pTrcMV 232 , and pTrcMK 234 , respectively. To create a restriction enzyme site (XbaI) at the 3’ end of the mipAt gene, two amino acids (Ser and Arg) were added at the C-terminus.
성능/효과
2. The results showed that lipase activity could be measured from all recombinant E. coli display strains to varying degrees, except for a control. Among the six mipA derivatives, MV 140 was the best display motif.
These results indicate that the MV140 motif could be used for successfully displaying another enzyme, α-amylase tagged with FLAG on the E. coli cell surface, but the previous motif, YiaTR232 (55 U/L), was a little more efficient as a display motif for α-amylase than the MV140 .
참고문헌 (31)
1 Lee SY Choi JH Xu Z 2003 Microbial cell-surface display Trends Biotechnol. 21 45 52 10.1016/S0167-7799(02)00006-9 12480350
3 Wu CH Mulchandani A Chen W 2008 Versatile microbial surface-display for environmental remediation and biofuels production Trends Microbiol. 16 181 188 10.1016/j.tim.2008.01.003 18321708
4 Kondo A Tanaka T Hasunuma T Ogino C 2010 Applications of yeast cell-surface display in bio-refinery Recent Pat. Biotechnol. 4 226 234 10.2174/187220810793611509 21171959
5 Kuroda K Ueda M 2011 Cell surface engineering of yeast for applications in white biotechnology Biotechnol. Lett. 33 1 9 10.1007/s10529-010-0403-9 20872167
6 Faber K Frassen MC 1993 Prospects for the increased application of biocatalysts in organic transformations Trends Biotechnol. 11 461 470 10.1016/0167-7799(93)90079-O 7764400
8 Lee SH Choi JI Park SJ Lee SY Park BC 2004 Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis Appl. Environ. Microbiol. 70 5074 5080 10.1128/AEM.70.9.5074-5080.2004 15345384
9 Lee SH Choi JI Han MJ Choi JH Lee SY 2005 Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent Biotechnol. Bioeng. 90 223 230 10.1002/bit.20399 15739170
10 Georgiou G Stathopoulos C Daugherty PS Nayak AR Iverson BL Curtiss RI 1997 Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines Nat. Biotechnol. 15 29 34 10.1038/nbt0197-29 9035102
11 Jung HC Lebeault JM Pan JG 1998 Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae Nat. Biotechnol. 16 576 580 10.1038/nbt0698-576 9624691
12 Xu Z Lee SY 1999 Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif Appl. Environ. Microbiol. 65 5142 5147 10.1128/AEM.65.11.5142-5147.1999 10543834
15 Ko KC Lee B Cheong DE Han Y Choi JH Song JJ 2015 Bacterial cell surface display of a multifunctional cellulolytic enzyme screened from a bovine rumen metagenomic resource J. Microbiol Biotechnol. 25 1835 1841 10.4014/jmb.1507.07030 26323268
16 Han MJ Lee SY Koh ST Noh SG Han WH 2010 Biotechnological applications of microbial proteomes J. Biotechnol. 145 341 349 10.1016/j.jbiotec.2009.12.018 20045032
17 Nhan NT Gonzalez de Valdivia E Gustavsson M Hai TN Larsson G 2011 Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus Microb. Cell Fact. 10 22 10.1186/1475-2859-10-22 21481238
19 Yim SS An SJ Han MJ Choi JW Jeong KJ 2013 Isolation of a potential anchoring motif based on proteome analysis of Escherichia coli and its use for cell surface display Appl. Biochem. Biotechnol. 170 787 804 10.1007/s12010-013-0236-9 23613117
20 Han MJ Lee SH 2015 An efficient bacterial surface display system based on a novel outer membrane anchoring element from the Escherichia coli protein YiaT FEMS Microbiol. Lett. 362 1 7 10.1093/femsle/fnu002 25790485
21 Sambrook J Russell DW 2001 Molecular Cloning: A Laboratory Manual 3rd Ed Cold Spring Harbor Laboratory Press New York
22 Han MJ Wang H Beer LA Tang HY Herlyn M Speicher DW 2010 A systems biology analysis of metastatic melanoma using in- depth three-dimensional protein profiling Proteomics 10 4450 4462 10.1002/pmic.200900549 21136598
23 Li H Zhang DF Lin XM Peng XX 2015 Outer membrane proteomics of kanamycin-resistant Escherichia coli identified MipA as a novel antibiotic resistance-related protein FEMS Microbiol. Lett. 362 1 8 10.1093/femsle/fnv074
24 Han MJ Lee SY Hong SH 2012 Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains J. Microbiol. Biotechnol. 22 470 478 10.4014/jmb.1110.10080 22534293
25 Molloy MP Herbert BR Slade MB Rabilloud T Nouwens AS Williams KL Gooley AA 2000 Proteomic analysis of the Escherichia coli outer membrane Eur. J. Biochem. 267 2871 2881 10.1046/j.1432-1327.2000.01296.x 10806384
26 Stenberg F Chovanec P Maslen SL Robinson CV Ilag LL von Heijne G 2005 Protein complexes of the Escherichia coli cell envelope J. Biol. Chem. 280 34409 34419 10.1074/jbc.M506479200 16079137
27 Bagos PG Liakopoulos TD Spyropoulos IC Hamodrakas SJ 2004 PRED-TMBB: a web server for predicting the topology of beta- barrel outer membrane proteins Nucleic Acids Res. 32 W400 W404 10.1093/nar/gkh417 15215419
29 Ahn JH Pan JG Rhee JS 1999 Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1 J. Bacteriol. 181 1847 1852 10.1128/JB.181.6.1847-1852.1999 10074078
31 Yang Z Liu Q Wang Q Zhang Y 2008 Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium Vibrio anguillarum Appl. Environ. Microbiol. 74 4359 4365 10.1128/AEM.02499-07 18487403
※ AI-Helper는 부적절한 답변을 할 수 있습니다.