The refolding of recombinant human liver methylmalonyl-CoA mutase from inclusion bodies produced in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
Loading...
Date
1998
DOI
Open Access Location
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Massey University
Rights
The Author
Abstract
Human methylmalonyl-CoA mutase (hMCM) is an adenosylcobalamin-dependent enzyme that catalyses the structural rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA as pan of the catabolism of the branched chain amino acids valine, leucine and isoleucine, odd chain fatty acids and intermediates of cholesterol metabolism. Reactions that require adenosylcobalamin (AdoCbl) have been intensively studied, and the first step in the catalysis is widely agreed to involve homolytic cleavage of the unusual carbon-cobalt bond in the cofactor. A reliable source of recombinant hMCM would be useful in defining more fully the mechanistic pathway of AdoCbl-dependent enzymes. Recombinant hMCM overexpressed in E. coli forms insoluble aggregates of inactive protein known as inclusion bodies. hMCM inclusion bodies were purified, solubilised and then several different in vitro refolding techniques were tested in attempts to produce active recombinant hMCM from purified solubilised inclusion body material. These methods included refolding by rapid dilution, refolding by dialysis, detergent-assisted refolding, refolding by gel filtration chromatography and chaperonin-assisted refolding. Chaperonin-assisted refolding necessitated the purification of recombinant E. coli chaperonins GroES and GroEL from the E. coli strain DH1/pGroESL. Refolding by rapid dilution of the GdmHCl-solubilised inclusion bodies into a refolding buffer was judged to be the simplest and most effective method, however the refolding process was extremely inefficient. Refolding by rapid dilution was scaled up to 2 litres to produce as much active hMCM as possible. The refolded protein was concentrated by batch adsorption to and stepwise elution from hydroxyapatite, and further purified using a synthesised 5'adenosylcobalamin- agarose 'affinity' chromatography column. The final refolded hMCM preparation contained a single ~29 kDa contaminant protein, tentatively identified as E. coli branched-chain amino acid aminotransferase (EC 2.6.1.42), present in approximately equal amounts to the hMCM, and had a specific activity of ~3.11 units/mg.
Description
Keywords
Escherichia coli, Enzymes -- Biotechnology, Recombinant proteins