Overcoming The Codon Bias Of E. Coli For Enhanced Heterologous Protein Expression

Escherichia coli uses different codons encoding the same amino acid with different frequencies,and those codons that are not frequently used are called rare codon.Rare codon can reduce the efficiency of protein expression.In order to improve the expression of heterologous proteins in E.coli BL21(DE3),in this experiment,the t RNA genes corresponding to the rare codons of E.coli BL21(DE3)have been assembled in a synthetic fragment and inserted into a ribosomal operon to construct the strain E.coli BL21(DE3)-rrn D::6t RNA,where the number of t RNAs can dynamically coordinate with the number of amino acids.The amount of Cbi A expressed by this strain is 1.49 times than E.coli BL21(DE3).This result lays the foundation for industrially overcoming the codon bias of E.coli to express heterologous proteins with high efficiency.The method of this experiment is pCas/pTargetF system.The vector pCas contains homologous recombination-related Exo,Beta,Gam proteins and Cas9 protein,which plays the role of cutting DNA double strands and homologous recombination.The vector pTargetF contains the N20 sequence,which plays the role of guiding Cas9 protein to cut DNA double strands.The 6 t RNA genes corresponding to the rare codons of E.coli BL21(DE3)were designed as a synthetic fragment,and the homology arms are connected to construct the target fragment.Target fragment and the vector pTargetF were cotransformed into the E.coli BL21(DE3)-pCas strain,the E.coli BL21(DE3)-rrn D::6t RNA strain was successfully constructed.The growth curve indicating that the increase of t RNA gene inserted did not destroy the stability of the intracellular environment of E.coli BL21(DE3).The vector pet28a-Cbi A transformed into E.coli BL21(DE3)and E.coli BL21(DE3)-rrn D::6t RNA strains.The optimal expression is obtained when the induction concentration of IPTG is 0.01 m M at 37? and the induction time is 4 h.SDS-PAGE and Western-Blot are used to quantitatively analyze the differences in the expression of heterologous proteins.The amount of Cbi A expressed by E.coli BL21(DE3)-rrn D::6t RNA is 1.49 times than E.coli BL21(DE3).