Improving Escherichia coli prokaryotic expression system with low virulence and high efficiency by recombination technology

Abstract:

[Background] As a common host used in prokaryotic expression system, Escherichia coli owned many advantages like low cultivation cost, short growth period and strong operability while also shared some deficiencies isochronally. [Objective] To decrease the endotoxin biosynthesis and virulence of E. coli and improve its exogenous proteins soluble expression ability synchronously. [Methods] lpxM gene in lipopolysaccharide biosynthesis pathway of E. coli was primarily deleted by CRISPR-Cas technology for lipid A side chain structure modification. Then tig gene was integrated expressed in genome of E. coli BL21(DE3) ?lpxM to provide chaperon factor for exogenous proteins. Besides, the recombinant plasmid pET-28a-Rcodon was constructed to supply the rare codon relevant tRNA for protein expression. [Results] Bacterial somatic endotoxin level obtained a 90% reduction compared to the original strain after lpxM deletion. By utilizing the recombinant host strain and plasmid, expression level of the infectious bursal disease virus (IBDV) VP2 protein was observably enhanced. Clinical safety evaluation results indicated that the endotoxin virulence of E. coli BL21(DE3) ?lpxM::tig was apparently lower than the original strain and the relevant immunity group showed no clinical symptoms. [Conclusion] E. coli prokaryotic expression system was remolded by recombinant technologies for low virulence and high soluble expression of the exogenous proteins, which laid a certain foundation for the relevant subunit vaccines investigation.