Study On Enhanced Secretion Of Soluble Green Fluorescent Protein In Escherichia Coli

Escherichia coli(E.coli),as one of the best-characterized host cells,possesses a short growth cycle,low cultural cost,and suitable for industrial production.It has been widely used as a cell factory for the production of heterologous proteins.The expression of heterologous proteins in E.coli can be divided into two typs,intracellular expression and extracellular secretion.The production strategy of extracellular secreted heterologous proteins avoids the cell lysis step in the protein extraction process,consquently,it is simpler and more cost-effective than intracellular production,and a preferred solution for engineering bacterial protein in industrial production.Protein secretion in E.coli is usually directed to specific secretory pathways under the guidance of signal peptides.However,there exist a no-defined secretory system,E.coli superfolder green fluorescent protein(sf GFP)mediated Protein Secretion System(EGPSS).sf GFP can guide the secretion of its fused protein to extracellular media and improve the solubility of the fusion protein without affecting the latter conformation and function.In order to further increase the autocrine level of highly soluble green fluorescent protein(sGFP)in E.coli,explore the potential effect of outer membrane protein of LPP in EGPSS secretion system,and provide a technical basis for augmenting extrocellular secretion of sGFP fused protein in later experiments,this study first replaced the auxiliary plasmid p KD46 promoter in Red recombinant system with heat shock promoter Phs,to eliminate the limitation of p KD46 plasmid in the process of E.coli Red recombination,that is,to simplify the approach and raise the accurate and effectivity of induced proteins expression essential for heterologous gene or DNA fragments integrating into E.coli chromosome in site-specifically.The modified Red recombinant system helper plasmid was defined as p KD-Phs.Then,the murein lipoprotein(lpp)gene encode E.coli outer membrane lipoprotein was knocked out by the modified Red recombinant system and replaced with sGFP-Kan expression cassette to create mutant strain of BL21(DE3)lpp::sGFP.Then,the optimal induction time of mutant strain BL21(DE3)lpp::sGFP and p ET29b-sGFP transformed strain in BL21(DE3)was determined.Then,at the optimal induction time,the level of sGFP secretion was analyzed to determine the effect of lpp gene knockout on sGFP secretion.The results showed that the heat shock promoter used in this study had high functional efficiency in E.coli,though its efficacy was slightly lower than that of T7 promoter,which could be used to modify Red recombinant helper plasmid.Through the knock-in with sGFPKan expression cassette and simultaneously knockout of lpp gene in E.coli,the mutant strain BL21(DE3)lpp::sGFP,was successfully obtained,which retained genetic stability.At the same time,the knockout of lpp gene had little effect on the growth cycle of E.coli,and the growth curve of mutant strain was similar to that of wild strain.The best IPTG induction time for sGFP expression both in mutant strain BL21(DE3)of lpp::sGFP and p ET29b-sGFP transformed strain of BL21(DE3)was 26 h and 28 h,respectively.The analysis of sGFP secretion level of mutant strain and transformed strain showed that the extracellular protein concentration of mutant strain sGFP accounted for 87 % of the total sGFP concentration,which was higher than that at 74 % of the sGFP transformed strain.In this paper,through the knock-in with sGFP-Kan expression cassette and simultaneously knockout at lpp gene site in E.coli chromosome,the following results were successfully obtained.Firstly,stabilized chromosomal site-specific knock-in and expression of sGFP in E.coli provided a visualized report gene's tag for chromosomal gene knock-in in E.coli.Secondly,the levels of sGFP in-situ expression and secreting to extracellular media were higher than those of sGFP transient expression in its transformed strain.Finally,lpp knock-out result preliminarily revealed that LPP protein could involve in the pathway of sGFP secreting to extracellular media,which provided a technical basis for further exploring the mechanism of EGPSS and increasing secretory level of sGFP's fusion proteins.