The Identification Of MDH And CysE Eliminylaton In Escherichia Coli

The eliminylation is a newly identified and uncommon protein post-translational modification,the thiol group can attach the unsaturated amino result in Michael covalent addition.In recent years,the eliminylation proteins had been identified were crystalline protein,MAPKs and Glutathione peroxidase.Conversely,the polypeptide had been identified more tow hundreds.For the identification of eliminylation proteins and polypeptides,there isn't the favorable approaches,therefore,to fill the gap which can identify the more eliminylation protein and polypeptide.We labeled the E.coli lysis based on the maturing labeling probe,and identified 221 proteins which maybe behave the eliminylation.And,we chosen two of them,including MDH and CysE,to make further identify.Firstly,we constructed recombinant proteins Pet28a-MDH and Pet28a-CysE,and transformed them to E.coli to achieve the affinity purification by using Ni-TED.Meanwhile,using the labeling probe of bio-SH to preliminary label the target proteins,and founding that the two proteins both had the superior effectivity of addition reaction,furthermore,for the non-specificity reaction maybe exit in the addition reaction and identifying the specific of the bio-SH probe,we utilized the DTT to compete which could avoid the non-specificity,and the two proteins show the obviously reduced in addition reaction.Secondly,we utilized the streptavidin immobilization beads to incubate with labeled proteins in room temperature,to detect the two proteins' column efficiency,this characterization will provide the preparation for further study.Finally,utilizing the CRISPR/Cas9 to knockout the MDH gene,and constructed their compensation strain to achieve the overexpression of target proteins.Comparing the addition efficiency of overexpressed proteins and non-overexpressed proteins,we found that the addition efficiency of overexpressed proteins were higher.This paper is based on our laboratory mature labeling probe.Specifically labeling the MDH and CysE in the condition of avoiding the non-specificity,and detecting the column efficiency of them,finally,through the construction of knockout system,we can obtain the theoretical basis for the identification of MDH and CysE eliminylation in E.coli.Moreover,this study also provide a theoretical basis for the further study of the identification of eliminylated proteins and peptides...