[Construction and expression of prokaryotic expression vector pTWIN1/TRAIL].
OBJECTIVE
To purify an active extracellular region of the TNF related apoptosis inducing ligand (TRAIL) protein.


METHODS
According to the high-usage codons in Escherichia coli and the multiple cloning site of expression vector pTWIN1 of a self-splicing prokaryotic expression system, the extracellular region of TRAIL gene was designed and synthesized, which was cloned into pMD18-T vector. After pMD18-T/TRAIL and pTWIN1 were digested by Nru I and EcoR I, the target fragment purified was linked to the expression vector pTWIN1, which was transferred into the competent cell JM109, and positive recombination was screened. After positive recombination vector pTWIN1/TRAIL (identified with restrictive endonuclease digesting and sequencing) was transferred into the ER2566, the expression was induced by different IPTG concentration at different temperature and culture time. The expression products were analyzed by 150 g/L SDS-PAGE.


RESULTS
The extracellular region of TRAIL gene was obtained by PCR, and was constructed successfully in a self-splicing prokaryotic expression vector pTWIN1/TRAIL. By 0.3 mmol/L IPTG at 15 degrees C for 14 to 16 hours, the soluble target protein was expressed efficiently.


CONCLUSIONS
High-expression level of the extracellular region of TRAIL fusion protein was attained by use of E. coli ER2566, and the soluble target protein without any additional amino acid was successfully purified by simple treatment.
Yang
Wen-li
WL
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Chen
Shou-hun
SH
Chen
Yi-rong
YR
Qin
Yang
Y
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000096:biosynthesis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand