Recombinant expression, purification and characterization of acetylated LysargiNase from Escherichia coli with high activity and stability

Abstract

Rationale: LysargiNase is a novel characterized metalloprotease that can cleave the N-terminii of lysine or arginine residues. The peptides generated by LysargiNase are just mirrors to those generated by trypsin. These characteristics of LysargiNase provide a powerful tool for mass spectrometry (MS)-based proteomics research. A highly active and stable LysargiNase produced by an easy and inexpensive method could greatly benefit proteomics research. Here, we report the soluble recombinant expression, purification and acetyl modification of LysargiNase in Escherichia coli.

Methods: The coding sequence of LysargiNase with an enterokinase cleavage site at the N-terminus was inserted into plasmid pGEX-4 T-2 and transformed into E. coli BL21 (DE3). The strain was cultured in a 14-L fermenter with a working volume of 5 L. The protein expression was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM. The recombinant LysargiNase was loaded onto a GSTrap and an on-column digestion was performed to remove the GST tag and was subsequently purified by chromatographic purification. In vitro acetylation of LysargiNase was performed by using acetic anhydride. The digestion efficiency and specificity of recombinant LysargiNase and acetylated LysargiNase were compared with simple protein substrate, human serum albumin (HSA), and a complex proteomic sample, yeast lysate, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS).

Results: Highly soluble expression of recombinant LysargiNase was achieved by plasmid pGEX-4 T-2 in E. coli BL21 (DE3). In addition, acetylation of purified LysargiNase significantly increased its resistance to autolysis, which resulted in a more complete digestion of proteomics samples and more identified peptides and proteins by LC/MS/MS.

Conclusions: In this study, we constructed a highly soluble expression system for producing recombinant LysargiNase in E. coli, which gave tremendous advantages in the downstream purification process. We also confirmed that acetyl modification can increase the stability and activity of recombinant LysargiNase. The study provided a superior way to produce this powerful tool for proteomics research.