[Construction of human PPARalphaLBD fusion protein expression vector and optimization of inducing conditions].
Peroxisome proliferator-activated receptoralpha (PPARalpha) is a ligand-activated transcription factor which plays a pivotal role in the regulations of metabolism. A cDNA encoding ligand binding domain (LBD) of PPARalpha was amplified by RT-PCR from human hepatic tissue and the product was inserted into the downstream of the malE gene in the vector pMAL-p2X,which encodes maltose-binding protein (MBP). The recombinant plasmid containing MBP-PPARalpha gene was transformed into E. coli. TB1, and then the growth conditions of the recombinant strain were studied, which remarkably influenced the final yield of protein expression. With the use of SDS-PAGE and Bio-Rad Quantity One gel image analysis, we found the best expression condition as follows: The induction was started as OD600 reached 0.5 by the adding of IPTG to a final concentration of 0.4 mmol/L, and then the incubation continued 6 hours at 30 degrees C. The maximum yield of fusion protein was 31.34% of the total mass of cytoplasm proteins. Moreover, the soluble form of the target protein is useful for further work on purification and on screening the ligands of PPARalpha.
Li
Changqing
C
Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China.
Zhang
Tao
T
Yuan
Ye
Y
Zhou
Qixin
Q
D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008024:Ligands; D058619:Maltose-Binding Proteins; D047493:PPAR alpha / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D047493:PPAR alpha; D011993:Recombinant Fusion Proteins