Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm

Ningjia He, Hiroshi Fujii, Takahiro Kusakabe, Yoichi Aso, Yutaka Banno, Kohji Yamamoto

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Present research provided an efficient approach to obtain large quantities of active recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm, Bombyx mori. The cDNA encoding mature CI-b1 was cloned into pDEST17 vector. Recombinant protein with hexa-histidine tag attached to the N-terminal of CI-b1 was expressed in Escherichia coli Origami B cells. It can be purified to homogeneity via the gel filtration chromatography on a Sephacryl S-200 column followed the affinity chromatography on a Ni-NTA column. The two sequential purification procedures yielded 4.3 mg purified (His) 6-tagged CI-b1 from 200 ml of culture medium. Studies on (His) 6-tagged CI-b1 revealed that three disulfide bonds were formed in the recombinant CI-b1 and the inhibitory properties of recombinant CI-b1 against α-chymotrypsin were similar to those of native CI-b1. Recombinant CI-b1 immobilized on Ni-NTA resin was used to detect the interactions occurring between the CI-b1 and its target factors.

Original languageEnglish
Pages (from-to)9-16
Number of pages8
JournalProtein Expression and Purification
Volume38
Issue number1
DOIs
Publication statusPublished - Nov 1 2004

All Science Journal Classification (ASJC) codes

  • Biotechnology

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