[Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity].
Sheng Wu Gong Cheng Xue Bao
; 24(7): 1194-8, 2008 Jul.
Article
en Zh
| MEDLINE
| ID: mdl-18837394
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-IdeltaD86:6His 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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Oryza
/
Proteínas Recombinantes de Fusión
/
Cistatinas
/
Inhibidores de Cisteína Proteinasa
/
Mutación
Idioma:
Zh
Revista:
Sheng Wu Gong Cheng Xue Bao
Asunto de la revista:
BIOTECNOLOGIA
Año:
2008
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
China