Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

Vol 15, Issue 4, 2013; Page No.(815-821)

HIGH-LEVEL SOLUBLE EXPRESSION OF BOVINE ENTEROKINASE CATALYTIC LIGHT CHAIN WITH NUS-TAG IN ESCHERICHIA COLI

MAHAMMAD AZHAR AND R. SOMASHEKHAR

Abstract

Enterokinase (E.C.3.4.21.9) acts as a sequence-specific protease. It activates trypsinogen by cleaving its inhibitory portion.Enterokinase has wide utility in cleaving fusion proteins. Bovine Enterokinase light chain (bEK L ) encoding nucleotide sequence was isolated from Bos taurus indicus (Gene Bank Accession No. KC756844) and one amino acid mutation (Proline121Serine) was identified. Bovine Enterokinase light chain is prone to form inclusion bodies when expressed inEscherichia coli (E. coli).The bEK L gene was inserted into pET21a, pET32a and pET43a expression vectors, and fused to T7 tag (The initial 11amono acids of the T7 gene10 protein), thioredoxin (Trx) tag and Nus tag (N-utilizing substance A) respectively .The recombinant bovine Enterokinase light chain (rEK L ) constructs were expressed in E.ColiRosetta strain, and it was observed that T7 -bEK L, Trx-bEK L expressing mainly as an inclusion body, but Nus- bEK L most of the fusion protein existed in soluble form. High level of bovine Enterokinase light chain-Nus tag protein production was achieved (200mg) from a 1litre culture. 120mg protein was isolated from a 1liter culture. After cleavage, 28 mg of pure active catalytic light chain was obtained. The rEK Lwas showing Kmvalue 0.65 mM and K cat 1500min -1 for fluorometric substrate Gly-(Asp ) 4 - Lys-β-naphthylamide. These values were found similar to previously published data for bovine EK light chain, and showed that mutation has no effect on its enzyme activity. Nus tag has increased EK L Solubilityand Expression levels.

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