Effect Of Molecular Chaperones And Fusion Tags On Improving Protein Folding Of The TEV Protease

The research on protein folding error caused by synonymous codon mutation has becoming a hotspot.The synonymous codon mutation in human gene can lead to some genetic disease.When increasing heterologous protein expression level by codon optimization in E.coli,some proteins such as tobacco etch virus protease show poor protein folding.The research on improving these synonymous codon variants' folding had rarely been reported.In this study,we used two groups of molecular chaperones and two fusion tags to find their effect on improving TEVp variants folding in E.coli.The results are shown as follows:1.GFP fused TEVp variants(three amino acid variants and two synonymous variants)were coexpressing with molecular chaperone group A(GroEL,GroES,GrpE)or group B(DnaK,DnaJ,ClpB)at 16? for 24 h.The effect of molecular chaperone Group B on improving TEVp variants' expression level is higher than group A.Under low temperature,the selected molecular chaperones showed effective on improving different variants folding.2.Analyzed the molecular chaperones effect on improving TEVp variants folding under heat shock condition by GFP qualitative and quantitative detection.Only molecular chaperone group A had improved the variants' expression level,group B had no effect at37?for 4h.Indicated that not all chaperones had effect under high temperature.3.Analyzed the effect of two fusion tags and TEVp recognition sequence on six TEVp variants' expression level.The recombinant MBP fused TEVp variants had been detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis obviously.The existence of TEVp recognition sequence had decreased MBP fused TEVp variants' soluble expression level.Trx fused TEVp variants' expression level had no obviously improved.4.Analyzed the activity of all four groups of tag fused.MBP fusion can improve the TEVp amino acid variants' activity.The TEVp recognition sequence between MBP and TEVp can further enhance this improvement.Trx fusion can also improving TEVp amino acid variants activity.But the improvement did not effect by TEVp recognition sequence.Neither MBP tag or Trx tag had effect on TEVp synonymous variants' activity improvement.5.Analyzed the activity of all four groups of tag fused TEVp variants expressed inRosettaTM(DE 3),which increased the rare tRNA abundance in E.coli.The activity of amino acid variants got high increased,the activity of synonymous variants did not improve either.In conclusion,we had prove that molecular chaperones do improve TEVp expression level,especially can help TEVp synonymous variants folding under low temperature.Different tags produced different effect on amino acid variants.In order to get a suitable tag for improving protein expression level two or more fusion tags should be scan.Fusion tags or tRNA abundance can hardly improve activity of the synonymous variants which suggests that the synonymous variants' folding damage is genetic controlled.It difficult to be repaired in the translation level.