Recombinant Expression And Purification Of Glutamyl Endopeptidase C And Its Application In Proteomics

Proteomics is one of the hot areas in the post-genome era which is one study of the components,functions and interactions of proteins.With the rapid development of mass spectrometry technology,proteomics research has also made great progress and has a bright future.There are three research strategies of proteomics:top-down,middle-down,and bottom-up strategies.The workflow of the bottom-up strategy is to digest the extracted protein samples to produce suitable lengths peptides and then identified by mass spectrometry and database matching analysis.For the specific digested peptides produced by this strategy are suitable for mass spectrometry detection,and the information obtained is more abundant,it has become the main technique of proteomics research.The enzymatic hydrolysis of protein samples has a huge impact on the results of mass spectrometry detection,The degree of enzymatic hydrolysis of protein samples determines the output efficiency of peptides and the quality of mass spectrometry,which can reduce the complexity of samples,improve the efficiency of mass spectrometry identification and improve the accuracy of quantification.so the development of site-specific and highly active tool enzymes commonly used in proteomics research is particularly important.Trypsin,which specifically cleaves peptide bonds at the carboxyl termini of lysine and arginine,is the most commonly used tool enzyme for proteomics research.However,with the deepening of proteomics research and the improvement of the requirements for the identification of proteomic samples which has different amino acid sequences,more and more proteases,such as lysyl endonuclease(Lys-C),aspartamido endonuclease(Asp-N),and glutamyl endonuclease C(Glu C)etc have been developed and applied.Among them,Glu C specifically cleaves peptide bonds at the carboxyl terminus of glutamic acid or aspartic acid residues of proteins,which is helpful for identifying proteins with few basic amino acids.It can aslo significantly improve the mass spectrometry detection,reliability and sequence coverage of protein identification by used with other proteases.However,the current commercialized glutamyl endopeptidase C(Glu C)mainly depends on extraction from Staphylococcus aureus or Bacillus subtilis strains.The yield is low and the commercial products are expensive which limited the use of this enzyme to some extent.There are some reported studies on the expression and purification of Glu C in recombinant expression systems of Escherichia coli,Bacillus subtilis,and Streptomyces lividans etc,but degradation and easy inclusion body formation have seriously affected the productivity and activity of the protease.We chose E.coli as host to express the mature peptide encoding gene of Staphylococcus aures glutamyl endopeptidase C after codon optimization.The ORF of glutamyl endopeptidase C was inserted into p GEX-4T-2 expression vector to generate p GEX-4T-2-Glu C,which was transformed into E.coli BL21(DE3)to obtain engineered strains.By optimizing the conditions for the induction of expression of recombinant proteins,a highly soluble expression of recombinant Glu C in E.coli was achieved.GST-tag affinity chromatography,anion exchange chromatography and gel filtration chromatography were combined to get purified Glu C.The activity was evaluated by using quantitative proteomics technology with pure bovine blood albumin(BSA)and yeast total cell lysates(Yeast TCL),respectively.The enzyme digestion efficiency,enzyme digestion specificity,identified protein coverage and the number of the identified proteins were systematically compared,which proved that our prepared Glu C has good activity.