[Construction and application of a new prokaryotic expression vector derivative of pBV220].
A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.
Zhu
Daxing
D
Department of Lung Cancer Surgery, Lung Cancer Institute, General Hospital, Tianjin Medical University, Tianjin 300052, China.
Wang
Yanping
Y
Yang
Xueqin
X
Zhu
Wen
W
Chen
Xiaohe
X
Sun
Zhilin
Z
Zhou
Qinghua
Q
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D054778:NM23 Nucleoside Diphosphate Kinases / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D054778:NM23 Nucleoside Diphosphate Kinases; D011993:Recombinant Fusion Proteins