Abstract
Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.
Highlights
Adipose tissue has been shown to play an important role in the regulation of body energy homeostasis and metabolism, lipid storage and as an endocrine organ
There is obvious correlation between plasma ADP levels and metabolic syndrome [2,3,4] and various studies have shown a noticeable decrease in ADP levels in patients with obesity, type II diabetes and with the accumulation of visceral adipose tissue [5,6,7]
In this study we examined the activity of recombinant adiponectin which was produced by P. pastoris (P-ADP) compared with that produced by E. coli (E-ADP)
Summary
Adipose tissue has been shown to play an important role in the regulation of body energy homeostasis and metabolism, lipid storage and as an endocrine organ. Compared to prokaryotic expression systems like Escherichia coli, the advantages of eukaryotic expression system such as the methylotrophic yeast Pichia pastoris are many. These advantages include an efficient recombinant protein secretion pathway for ease of purification, the availability of eukaryotic post-translational modifications, fast growth on economic salt-based media and little risk of contamination with endo-toxins or oncogenic or viral DNAs [9]. The availability of strong and tightly regulated promoters makes this yeast a very attractive host for recombinant protein production [10]
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