Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1194-1198, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-275404
ABSTRACT
According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD866His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Oligonucleotides
/
Prokaryotic Cells
/
Oryza
/
Recombinant Fusion Proteins
/
Cysteine Endopeptidases
/
Papain
/
Cystatins
/
Cysteine Proteinase Inhibitors
/
Cloning, Molecular
/
Genes, Plant
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2008
Type:
Article
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