Construction of human PPARalphaLBD fusion protein expression vector and optimization of inducing conditions / 生物医学工程学杂志
Journal of Biomedical Engineering
;
(6): 908-916, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-342717
ABSTRACT
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Fusion Proteins
/
Carrier Proteins
/
PPAR alpha
/
Electrophoresis, Polyacrylamide Gel
/
Escherichia coli
/
Maltose-Binding Proteins
/
Genetic Vectors
/
Genetics
/
Ligands
/
Metabolism
Limits:
Humans
Language:
Chinese
Journal:
Journal of Biomedical Engineering
Year:
2008
Type:
Article
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