Expression and Purification of the Maturation Enzymes of the Astexin Lasso Peptides

Abstract

Lasso peptides are small natural products which are posttranslationally modified into a unique lasso shape that provides them with remarkable stability and pharmacologically relevant bioactivity. Of the known lasso peptides microcin J25 (MccJ25) has been extensively studied and its mode of action and biosynthesis have been characterized. The enzymes that give MccJ25 its final shape are McjB and McjC, and homologs of these enzymes exist for the maturation of most lasso peptides. However, most characterization studies have been conducted in vivo, and there are only a few studies that have attempted to purify the maturation enzymes of MccJ25 and other lasso peptides for in vitro studies of their activity and for structure determination. The goal for the present study was to purify the maturation enzymes of the astexin lasso peptides. We report the successful purification of the McjB homolog AtxB1, partly responsible for the maturation of astexin-1. AtxB1 was purified with a hexahistidine tag (His6) and as a fusion to MBP under denaturing and native conditions. A comparison between both methods was made and preliminary results suggest that even though the native conditions procedure is less cumbersome, the quality of the protein purified under denaturing conditions and refolded through dialysis is superior. We also report successful cleavage of the MBP soluble partner, as well as some insights regarding co-eluted contaminants. The McjB homolog, AtxB2, partly responsible for the maturation of astexin-2 and 3, was expressed as a recombinant protein with a His6 tag under denaturing conditions. Subsequent purification revealed that expression levels were low and that refolding through dialysis was not straightforward. Nonetheless, we regard the present study as a successful first step in purifying the astexin maturation enzymes. In vitro studies with AtxB1-2, and the McjC homologs AtxC1-2, could help refine our understanding of the biosynthesis of astexins-1, 2, 3 and other lasso peptides.