title: An efficient bacterial surface display system based on a novel outer membrane anchoring element from the Escherichia coli protein YiaT.
abstract: In a bacterial surface display system, the display of a successful recombinant protein is highly dependent on the choice of anchoring motif. In this study, we developed an efficient Escherichia coli display system using novel anchoring motifs derived from the protein YiaT. To determine the best surface-anchoring motif, full-length YiaT and two of its C-terminal truncated forms, cut at the R181 and R232 sites, were evaluated. Two industrial enzymes, a lipase from Pseudomonas fluorescens SIK W1 and an α-amylase from Bacillus subtilis, were used as the target proteins for display. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, immunofluorescence microscopy and whole-cell enzyme activity measurements confirmed the expression of the fusion proteins on the E. coli surface. Using YiaTR181 or YiaTR232 as the anchoring motif, the fusion proteins showed very high enzyme activities and did not exert any adverse effects on either cell growth or the outer membrane integrity. Additionally, these fusion proteins were suitable for displaying proteins of large molecular size in an active form. Compared with the previous anchoring motifs FadL and OprF, YiaTR181 and YiaTR232 had approximately 10-fold and 20-fold higher enzyme activities, respectively. These results suggest that YiaT can be used as an E. coli anchoring motif to efficiently display various enzymes; hence, this system could be employed in a variety of biotechnological and industrial applications.
authors: Department of Biomolecular and Chemical Engineering, Dongyang University, 145 Dongyang-daero, Punggi-eup, Yeongju, Gyeongbuk 750-711, Republic of Korea mjhan75@dyu.ac.kr.
authors: Mee-Jung
authors: MJ
authors: Han
authors: Industrial Biochemicals Research Group, Research Center for Biobased Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors: Seung Hwan
authors: SH
authors: Lee
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D015153:Blotting, Western; D061505:Cell Surface Display Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D008049:Lipase; D000516:alpha-Amylases
keywords: Escherichia coli; YiaT; cell surface display; outer membrane protein