A novel bacterial cell-surface display system based on NCgl1221 from Corynebacterium glutamicum

Abstract:

Abstract:［Objective］To develop a novel Escherichia coli cell surface display system by using C-terminally truncated NCgl1221 as the anchoring protein，which greatly enriched or optimized the bacterial displayed systems．［Methods］We amplified the sequence of C-terminally truncated NCgl1221 and β-amylase，and constructed the fusion expression vector．Then we transformed the recombinant plasmids PET-NA and PET-28a into Rosetta (DE3) pLysS．The fusion protein expression was induced by IPTG and identified by SDS-PAGE and Western blot analysis．The IPTG induced strains were immunostained and investigated by fluorescence microscope and flow cytometry to detect the displayed β-amylase．Finally， we analyzed the activity of β-amylase and starch hydrolization in order to determine whether the displayed β-amylase has the activity or not． ［Results］The fusion protein was successfully expressed in E． coli，and the active β-amylase was displayed on the cell surface by fusing it to the C terminus of the anchor． The recombinant strain displaying β-amylase can utilize soluble starch in the medium．［Conclusion］A novel E． coli surface display system by using C-terminally truncated NCgl1221 as the anchor motif was successfully developed． The active enzyme with a molecular size of 56 kDa was displayed on E． coli by this system，which provided the basis for the application of the system in whole-cell biocatalyst or biosorbent．

Key words:Keywords: Corynebacterium glutamicum，β-amylase，cell-surface display，NCgl1221