Chaperone-assisted overexpression of an active D-carbamoylase from Agrobacterium tumefaciens AM 10.

Sareen, D and Sharma, R and Vohra, R M (2001) Chaperone-assisted overexpression of an active D-carbamoylase from Agrobacterium tumefaciens AM 10. Protein expression and purification, 23 (3). pp. 374-9. ISSN 1046-5928

PDF vohra2001.pdf - Published Version Restricted to Registered users only Download (118Kb) | Request a copy

Abstract

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.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Science.
Subjects:	Q Science > QR Microbiology
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	05 Jan 2012 15:16
Last Modified:	05 Jan 2012 15:16
URI:	http://crdd.osdd.net/open/id/eprint/282

Actions (login required)

View Item