Enhancement Of Exogenous Protein Expression Based On Amino Acid Composition Of Escherichia Coli

The demand for large-scale protein production with genetic engineering has been rising in recent years,especially in the field of prokaryotic expression using engineered strains,and thus the study of improving exogenous protein expression has been one of the popular research themes.As translation efficiency-related studies keep advancing,the factors considered in translation efficiency models are expanding,mainly including codon order,local t RNA abundance,the regulation of t RNA gene expression,and the needs for ribosomes,m RNA structure and energy consumption of peptide chain folding.Among them,studies on t RNA availability are more widespread.During translation,t RNA can be regarded as a tool for transporting raw amino acids,and the higher co-adaptive relationship between them facilitates efficient protein synthesis.Therefore,our group speculates that the levels of t RNA genes and corresponding amino acids should be well matched to synthesize proteins more efficiently.Such a congruence would maximize translation efficiency and at the same time minimize resource/energy costs.And the TAAI(t RNA Gene Copy Number and Amino Acid Usage Accordance Index)is proposed for the co-adaptation between t RNA gene copy number and amino acid composition at the organismal scale.In this study,the TAAI was used as a theoretical guideline to determine the adaptation index between t RNA gene copy number and amino acid usage accordance index of E.coli genome,and Gro EL,a molecular chaperone protein from Archaea,was selected as the exogenous protein with E.coli BL21(DE3)as the expression host bacterium to verify the increase of exogenous protein expression by manipulating the copy number of E.coli t RNA gene.Based on the results of TAAI calculation of E.coli genome,seven types of t RNA genes were selected,ile U,gly X,ala W,val V,glu W,asp U and ser V,which could increase TAAI by increasing copy number,and plasmid p ACYC-Duet1 was selected to construct seven recombinant vectors for application in exogenous protein expression studies.Then we selected p ACYC-Duet1 vector,vector inserted with t RNA gene copy asp U and vector inserted with seven t RNA gene synthetic fragment PT7 t RNA,respectively,for exogenous protein induction expression experiments and analyzed the protein expression differences.The results showed that the exogenous protein expression of vectors inserted with single copy of t RNA gene was higher than that of the empty vector,but the variance was not significant;the exogenous protein expression of vectors inserted with PT7 t RNA fragment was significantly higher than that of the empty vector,and the amount of improvement reached 21%.The common engineered strains or eukaryotic host cells used for exogenous protein expression were collected for relevant TAAI calculation analysis,with a view to provide references and help for protein commercial production.In this study,we found that the genomic TAAI of prokaryotic species increased with the addition of one t RNA gene copy corresponding to amino acid Ala,while the genomic TAAI of species decreased with the addition of one t RNA gene copy corresponding to amino acids Asn,Tyr and Cys.The change pattern of genomic TAAI in eukaryotic species is not obvious yet.