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Construction of a cell‐surface display system based on the N‐terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins
Author(s) -
Bao S.,
Yu S.,
Guo X.,
Zhang F.,
Sun Y.,
Tan L.,
Duan Y.,
Lu F.,
Qiu X.,
Ding C.
Publication year - 2015
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12824
Subject(s) - adhesion , domain (mathematical analysis) , identification (biology) , terminal (telecommunication) , nucleation , microbiology and biotechnology , cell adhesion , biology , chemistry , cell , biochemistry , computer science , ecology , mathematical analysis , telecommunications , mathematics , organic chemistry
Aims To construct and demonstrate a surface display system that could be used to identify mycoplasma adhesion proteins. Methods and Results Using the N‐terminal domain of InaZ (Ina ZN ) as the anchoring motif and the enhanced green fluorescent protein ( EGFP ) as the reporter, the surface display system pET ‐Ina ZN ‐ EGFP was constructed. Then, the mgc2 gene which encodes an adhesin and the holB gene which encodes DNA polymerase III subunit delta′ (nonadhesin, negative control) of Mycoplasma gallisepticum were cloned into the pET ‐Ina ZN ‐ EGFP respectively. The fusion proteins were expressed in Escherichia coli BL 21 ( DE 3). The distribution of the fusion proteins in E. coli cells was determined using SDS ‐ PAGE followed by Western blotting, based on cell fractionation. Escherichia coli cell surface display of the fusion protein was confirmed by immunofluorescence microscopy. The results indicated that the fusion proteins were not only anchored to the outer membrane fraction but also were successfully displayed on the surface of E. coli cells. Adhesion analysis of E. coli harbouring Ina ZN ‐ EGFP ‐mgc2 to host cells showed that the MGC 2‐positive E. coli cells can effectively adhere to the surfaces of DF ‐1 cells. Conclusions A surface display system using the Ina ZN as the anchoring motif and EGFP as the reporter was developed to identify putative adhesins of mycoplasma . Results indicated that adhesion by the cytadhesin‐like protein MGC 2 of mycoplasma can be reproduced using this surface display system. Significance and Impact of the Study This is the first construction of surface display system which could be used to identify the adhesion proteins of mycoplasma . The method developed in this study can even be used to select and identify the adhesion proteins of other pathogens.