%A D Sareen
%A R Sharma
%A R M Vohra
%O Copyright of this article belongs to Elsevier Science.
%J Protein expression and purification
%T Chaperone-assisted overexpression of an active D-carbamoylase from Agrobacterium tumefaciens AM 10.
%X The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 was cloned by polymerase chain reaction in plasmid pET28a and was overexpressed in Escherichia coli JM109 (DE3). However, almost 80% of the enzyme remained trapped in inclusion bodies. To facilitate the expression of the properly folded active enzyme, the chaperones GroEL/ES were coexpressed in plasmid pKY206. This resulted in a 43-fold increase in active enzyme production compared to the wild-type strain. The histidyl-tagged D-carbamoylase was purified by a single step nickel-affinity chromatography to a specific activity of 9.5 U/mg protein.
%N 3
%P 374-9
%V 23
%D 2001
%I Elsevier Science
%L open282