TY - JOUR
T1 - Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm
AU - He, Ningjia
AU - Fujii, Hiroshi
AU - Kusakabe, Takahiro
AU - Aso, Yoichi
AU - Banno, Yutaka
AU - Yamamoto, Kohji
N1 - Funding Information:
This work is supported by National Bio-Resources project (Silkworm) of the Ministry of Education, Culture, Sports, Science and Technology of Japan and by project entitled on ‘Development of effective animal genome analysis techniques and the applications of useful genes’ of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
PY - 2004/11
Y1 - 2004/11
N2 - 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.
AB - 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.
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U2 - 10.1016/j.pep.2004.07.010
DO - 10.1016/j.pep.2004.07.010
M3 - Article
C2 - 15477076
AN - SCOPUS:5344266791
SN - 1046-5928
VL - 38
SP - 9
EP - 16
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -