[Expression of a new cloned nitroreductase gene NOR1 and purification of expressed product].
OBJECTIVE
NOR(1)is a good candidate of tumor suppressor/susceptibility gene associated with nasopharyngeal carcinoma. This study was designed to construct the prokaryotic expression vector and to investigate the expression of nitroreductase gene NOR(1)in Escherichia coli and to purify expressed product.


METHODS
Total RNA was subtracted from normal nasopharyngeal carcinoma tissue. The full length of NOR(1)gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHIand XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI,then the NOR(1)gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR(1)was identified by sequencing and digested with restriction enzymes. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express GST fusion protein. The result was confirmed by Western blot analysis and the purified targeted protein was obtained by affinity chromatography.


RESULTS
The 1.25kb NOR(1)gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). It existed not only in supernatant but also in precipitation of broken bacteria. The result was confirmed by Western blot analysis,and the purified targeted protein was obtained by affinity chromatography.


CONCLUSIONS
The successes in construction of expression vector of NOR(1), expression and purification of GST/NOR(1)fusion protein make it possible to prepare for the polyantibodies for NOR(1).
Nie
Xin-Min
XM
Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan, 410078, PR China.
Xiao
Bing-Yi
BY
Li
Xiao-Ling
XL
Zhang
Bi-Cheng
BC
Li
Wei-Fang
WF
Wang
Rong
R
Cao
Li
L
Li
Gui-Yuan
GY
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009601:Nitroreductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D009601:Nitroreductases