Establishment And Characterization Of Bacterial Cell Surface Display Systems Using AcmA Of Lactococcus Lactis As The Anchoring Motif

For functionally surface display of foreign proteins,in this study,the full-length and the N-terminal domain of a N-Acetylmuraminidase AcmA from Lactococcus lactis,were respectively employed as the anchoring motif to immobilize the heterologous proteins on the cell surface of L.lactis or Escherichia coli,and the surface display efficiencies were preliminarily investigated.The N-Acetylmuraminidase encoding gene acmA was initially generated by PCR manipulation from L.lactis MB191,a wild-type strain that was newly isolated and identified in this laboratory,followed by sequencing and construction of a chimeric gene of "acmA-gfp" through a C-terminal fusion strategy at which the green fluorescent gene (gfp) was positioned in the direct downstream of acmA in a continuous encoding frame. The recombinant plasmid pMB137 was obtained by ligating the chimeric gene of acmA-gfp into pMG36k,a derivative of the previous E.coli-L,lactis shuttle expression vector pMG36e by insertion of an additional kanamycin resistance gene,and a recombinant L.lactis strain MB137 was derived by transferring pMB137 into a L.lactis wild-type strain AS1.2829.SDS-PAGE analysis confirmed that the AcmA-GFP fusion protein was expressed with the predicted size of 74 kD in the recombinant L.lactis cells. Flow cytometry analysis of the immuno-labelled intact cells further confirmed that GFP was immobilized on the outer membrane.Furthermore,a 672 bp of endo-beta-l,3-1,4-glucanase encoding gene gls that was PCR-amplified from Bacillus sublitis BF7658,was used to substitute the gfp gene in the recombinant plasmid pMB137, and the recombinant plasmid pMB138 was thus obtained,followed by transferring pMB 138 into L.lactis AS1.2829 to generate the recombinant strain MB 138.As a result,a whole-cell catalytic capacity of glucanase in MB138 was found with a relative glucanae activity of 12 U/mL.In view of the fact that the N-terminal domain of AcmA was structurally similar to that of ice nucleation protein(INP),a previously demonstrated anchoring motif in Pseudomonas syringae and has been widely used in a variety of Gram-negative strains,to evaluate if the Gram-positive bacteria-derived AcmA could be also functional in Gram-negative bacteria systems,the N-terminal of acmA was PCR-amplified,and was used to construct a fusion gene of acmAn/gfp.Similarly,the full-length acmA was also used to construct the fusion gene of acmA/gfp.The fusion gene acmAn/gfp and acmA/gfp was inserted into the E.coli expression vector pTrcHis-C for construction of recombinant plasmid pMB 131 and pMB 132,respectively,and two recombinant strains MB 131 and MB132 were further obtained by transforming pMB131 and pMB132 into the E.coli recipient JM109.The surface immobilization the expressed fusion protein in the recombinant E.coli cells were multiply verfied by immunofluorescence microscopic examination,pronase accessibility and SDS sensitivity assays,western blot analysis of the subcellular membrane fractions,and FACS assays of immuno-labelled intact cells.On the basis of this,the recombinant strains harboring pMB 133 and pMB134 were constructed by substitution of the gfp gene in the pMB131 and pMB132 with gls gene.By the inducible expression of IPTG,the recombinant E.coli strains expressed the acmAn/gls or acmA/gls fusion genes,showed an apparent whole-cell glucanase activity compared to the control strain.To my knowledge,this is the first report that demonstrated the truncated N-terminal domain of AcmA could serve as the functional anchoring motif to direct the cell surface immobilization of the foreign protein in E.coli strain.