Expression of recombinant EGFP viasurface display of ice nucleation protein and TEV protease cleavage site

The cell-surface display is the expression of proteinson the surface of prokaryotic and eukaryotic cells. Several bacteria have cell surface-anchoring proteins such as ice nucleation protein (INP) that can be displaying passengers on the outer membrane. INP is an outer membrane protein, have N-terminal domain, which is important for linkage to the outer membrane. Green fluorescent protein (GFP) is a protein that fluorescence green in the presence of light and used in a variety of applications to study the organization and function of living systems. Enhanced GFP (EGFP) is one of the brightest variants of GFP. In this study, we expressed a gene construct in the form of InaK-N (atruncated form of Inak variant of INP)-TEV protease cleavage site-EGFP on the surface of E. coli BL21 (DE3) and JM109 (DE3) as host strains.The pallet of induced E.coli was collected by centrifuging and resuspended in the TEV protease reaction buffer. After incubation with TEV protease, a 27 KD EGFP band was observed in SDS–PAGE. To estimate the level of surface expression ([surface EGFP]/[total EGFP]), we recorded the fluorescence intensity of EGFP in intact E. coli cell before and after TEV cleavage reaction and then calculated the concentration of EGFP with standard plot using purified cytoplasmic EGFP. The requirement of cell disruption and the chromatographic process is totally omitted and proteins prone to accumulation as inclusion bodies and have disulfide bonds could be successfully expressed using this strategy.

کلیدواژه ها:

Bacterial surface display ، INP ، EGFP