EXPRESSION VECTORS FOR AFFINITY PURIFICATION AND RADIOLABELING OF PROTEINS USING ESCHERICHIA-COLI AS HOST

被引:65
作者
CHEN, BPC
HAI, TW
机构
[1] OHIO STATE UNIV,OHIO STATE BIOCHEM PROGRAM,COLUMBUS,OH 43210
[2] OHIO STATE UNIV,DEPT MED BIOCHEM,COLUMBUS,OH 43210
[3] OHIO STATE UNIV,OHIO STATE BIOTECHNOL CTR,COLUMBUS,OH 43210
关键词
HISTIDINE TAG; NICKEL-CHELATING COLUMN; HEART MUSCLE KINASE; PROTEIN PHOSPHORYLATION; T7 EXPRESSION SYSTEM;
D O I
10.1016/0378-1119(94)90525-8
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
We have constructed two convenient vectors to produce foreign proteins in Escherichia coli. The first vector was developed to produce histidine (His)-tagged fusion proteins. In addition to encoding six contiguous His residues, it contains three unique restriction sites that allow cloning of a blunt-ended DNA fragment in three different reading frames. Therefore, one can clone any gene of interest in this vector to make a fusion protein tagged with six His at the N terminus. The His-tag allows purification of the fusion protein to almost homogeneity by a nickel-chelating column in a single step. The second vector is a derivative of the first vector; it encodes two tandem phosphorylation sites for heart muscle kinase (HMK) immediately downstream from the His residues. Therefore, the resulting fusion protein can be radiolabeled using [gamma-P-32]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.
引用
收藏
页码:73 / 75
页数:3
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