Chaperone-assisted overexpression of an active D-carbamoylase from Agrobacterium tumefaciens AM 10.
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.
Sareen
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Institute of Microbial Technology, Sector 39A, Chandigarh, 160036, India.
Sharma
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Vohra
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RM
D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018835:Chaperonin 10 / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D002479:Inclusion Bodies / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D014169:Transformation, Bacterial
D018835:Chaperonin 10; D018834:Chaperonin 60; D011994:Recombinant Proteins; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase