Expressed Protein Ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase: an Application to a Protein Expressed as an Inclusion Body

Abstract

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted to address various biological questions. EPL of the 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, was attempted. First, EPSP synthase was split into the N- and C-terminal fragments, based upon the cysteine scanning mutagenesis. Each domain was fused to the intein proteins. The N-terminal fragment (EPSPSN, 1-237) was purified from the intein-fused protein with a thioester tag exposed at its C-terminus. The C-terminal fragment (EPSPSCCYS, residues 238CYS-427) expressed as an inclusion body, was purified from a strain coexpressing the C-terminal fragment (Met-238CYS-427) and an additional methionine aminopeptidase (MAP) which enhances the removal of the initiation methionine from the C-terminal fragment. The purified fragments were mixed to initiate ligation in the presence of 2 % thiophenol. The ligation efficiency was ~85 %. The ligated full-length EPSP synthase was refolded and further purified using Mono Q anion exchange column. Circular dichroism (CD) and enzyme activity assays showed that the ligated full-length EPSP synthase has ~35 % specific activity compared to that of wild-type EPSP synthase.