Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization.
The adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects. Adiponectin serum concentrations are in the microgram per milliliter range in healthy humans and inversely correlate with obesity and metabolic disorders. Accordingly, raising circulating adiponectin levels by direct administration may be an intriguing strategy in the treatment of obesity-related metabolic disorders. However production of large amounts of recombinant adiponectin protein is a primary obstacle so far. Here, we report a novel method for large amount production of globular adiponectin from E. coli inclusion bodies utilizing an alkaline-shock solubilization method without chaotropic agents followed by precipitation of the readily renaturing protein. Precipitation of the mildly solubilized protein capitalizes on advantages of inclusion body formation. This approach of inclusion body protein recovery provides access to gram scale amounts of globular adiponectin with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralize the pH and renature the protein, thus saving chemicals and time. The precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in cultured MCF7 cells and significantly lowered blood glucose levels in mice with streptozotocin induced type 1 diabetes.
Heiker
John T
JT
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Leipzig University, Brüderstrasse 34, 04103 Leipzig, Germany.
Klöting
Nora
N
Blüher
Matthias
M
Beck-Sickinger
Annette G
AG
D052242:Adiponectin / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D000468:Alkalies / Q000737:chemistry; D000818:Animals; D001786:Blood Glucose / Q000187:drug effects; D045744:Cell Line, Tumor; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D012995:Solubility; D014508:Urea / Q000737:chemistry
D052242:Adiponectin; D000468:Alkalies; D001786:Blood Glucose; D011994:Recombinant Proteins; D014508:Urea