| Based on the structural analysis of Escherichia coli (E. coli) outer membrane protein C (OmpC),a novel bacterial cell surface display system was established in this study. Heterologous proteins were fused into the exposure sites of OmpC,which is used as a carrier protein. This is the first example of cell surface display by using E. coli OmpC as a carrier. Polyhistidine (polyHis) peptide and xylanase were used as passengers to probe the possibilities of developing whole cell absorbent and whole cell catalyst.The primary structure of OmpC was analyzed and its folding model on outer membrane was predicted. The Pstl unique site and the î–©oRV unique site on external loop 7 (L7) were selected to be fused with heterologous sequences. The entire ompC gene with its promoter was cloned from the chromosomal DNA of E. coli K-12 strain MC4100 by PCR. One set of 6His was synthesized artificially by overlap PCR,and sets of 6His were ligated in tandem to generate (6His)2,(6His)3,(6His)6,(6His)12,and (6His)18. Sequences of (6His)n were inserted into the unique Pstl site to form ompC-(6His)n fusion genes. These fusion genes were subsequently introduced into host strain MC4100 to construct MC4100(pTCHP) recombinant strains. SDS-PAGE profiles suggested that all fusion proteins except OmpC-(6His)18 were expressed with full length. When cells were cultivated and induced at 30C,the expression levels were 34.7%,33.1%,32.8% and 26.7% of the total outer membrane proteins for OmpC-(6His),,OmpC-(6His)2,OmpC-(6His)3,and OmpC-(6His)6,respectively. When cells were cultivated and induced at 25C,the expression levels were 29.9% and 10.2% of the total outer membrane proteins for OmpC-(6His)6 and OmpC-(6His)12,respectively. Ni-NTA-agarose beads binding test showed that all MC4100(pTCHP) strains could attach to agarose beads expect MC4100(pTCHP18). In another word,polyHis peptides,such as (6His)1,(6His)2,(6His)3,(6His)6,and (6His),2 could be successfully displayed on the cell surface of recombinant E. coli strains. Cadmium adsorption test revealed that all tested recombinant strains had improved Cd2+ adsorption abilities. Among them,MC4100(pTCHP6) could absorb 32.0umol Cd2+ per gram (dry weight) cell weight,as 3-fold high as that of control strain. Physiological studies suggested that all MC4100(pTCHP) strains except MC4100(pTCHP2) were sensitive to SDS and EDTA. We thus assumed that the stability of outer membrane and the optimal display results might essentially come from the ionic compatibility of inserts and target loops.To avoid the possible competition between p-lactamase and OmpC-(6His)n fusionproteins for similar secretion pathway,the ampicillin resistance gene on pTCHP6 and pTCHP12 were substituted with kanamycin resistance gene. Recombinant plasmids pKCH6 and pKCH12 as well as recombinant strains MC4100(pKCH6) and MC4100(pKCH12) were consequently constructed. SDS-PAGE profiles suggested that the substitution failed to contribute the expression level of fusion proteins,p-lactamase might facilitate the display of fusion proteins instead. Two other pathways dealing with the extra proteins in periplsma,"attenuation model" and "transportation model",were therefore proposed.Xylanase structure gene was subcloned from pKJX4 by PCR,and was used to replace the sequence between Pstl site and EcoRV site on ompC to construct ompC-xyl fusion gene. Xylanase activity assay showed that enzyme activities of the TGI recombinant strains were one order higher than that of the MC4100 recombinant strains. SDS-PAGE profiles showed that only degraded fragments could be detected from the outer membrane of recombinant strains when TGI was used as a host cell,while both degraded fragments and intact proteins were detectable when MC4100 was used as a host cell,implying the functional incompatibility of OmpC and xylanase within OmpC-xylanase fusion proteins. Xylanse is the largest protein (185 amino acids) accepted by OmpC in this study,and is also one of the longest among analogous systems.
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