Functional expression of the globular domain of human adiponectin in Pichia pastoris.
Adiponectin is an adipokine that predominantly synthesized and secreted from adipocytes mainly in the white adipose tissue. Here, we report that we have successfully expressed human gAdiponectin (the globular domain of adiponectin) in the methylotrophic yeast Pichia pastoris after codon optimization and established the purification procedure. The human gAdiponectin gene was designed and synthesized by PCR according to the P. pastoris preferred codons, and then inserted into the P. pastoris pPIC9K expression vector. The plasmid was electroporated into the P. pastoris strain GS115 and only the G418 resistance colonies could produce the gAdiponectin. After fermentation and purification, we could get 1.2g of recombinant gAdiponectin (purity is approximately 95%) from a 24 L culture media. The recombinant gAdiponectin is fully functional as evidenced by induction the phosphorylation of ACC in differentiated C2C12 myotubes, significantly lowering the blood glucose level and accelerating the clearance of free fatty acid in animal models.
Liu
De-Guo
DG
Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical School, Fudan University, PO Box 238, 138 Yi Xue Yuan Road, Shanghai 200032, PR China.
Liu
Hong-Lei
HL
Song
Tan-Jing
TJ
Huang
Hai-Yan
HY
Li
Xi
X
Tang
Qi-Qun
QQ
D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D052242:Adiponectin / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001786:Blood Glucose / Q000378:metabolism; D002460:Cell Line; D003062:Codon / Q000235:genetics; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000473:pathology / Q000517:prevention & control; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000473:pathology / Q000517:prevention & control; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000097:blood; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D032446:Myoblasts / Q000166:cytology / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D052242:Adiponectin; D001786:Blood Glucose; D003062:Codon; D005227:Fatty Acids; D011994:Recombinant Proteins; D000103:Acetyl-CoA Carboxylase