Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli
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In order to facilitate the large-scale preparation of active class II polyhydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative that contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpression of this phaC1 gene under control of the strong Ø10 promoter was achieved in Escherichia coli BL21(DE3). The fusion protein was deposited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30% of total protein. The inclusion bodies were purified by selective solubilization, resulting in approx. 70–80% pure PHA synthase, then dissolved and denatured by 6M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni2+-nitrilotriacetate–agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the chaotropic reagent. This procedure avoided the aggregation of folding intermediates which often decreases the efficiency of refolding experiments. Finally, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8m-units/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which corresponds to 27% of the maximum specific activity of the native enzyme. The refolding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the spectrum resulted in the following secondary structure prediction: 10% α-helix, 50% β-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.
Biochemistry and Cell Biology not elsewhere classified