Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli

In order to facilitate the large-scale preparation of active class II polyh ydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative th at contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpres sion of this phaC1 gene under control of the strong circle divide 10 promot er was achieved in Escherichia coli BL21 (DE3). The fusion protein was depo sited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30 % of total protein. The inclusion bodies were purified by select ive solubilization, resulting in approx. 70-80% pure PHA synthase, then dis solved and denatured by 6 M guanidine hydrochloride. The denatured PHA synt hase was reversibly immobilized on a Ni2+-nitrilotriacetate-agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the cha otropic reagent. This procedure avoided the aggregation of folding intermed iates which often decreases the efficiency of refolding experiments. Finall y, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8 m-u nits/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which correspon ds to 270, of the maximum specific activity of the native enzyme. The refol ding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the s pectrum resulted in the following secondary structure prediction: 10 % alph a -helix, 50 % beta -sheet and 40% random coil. Get filtration chromatograp hy indicated an apparent molecular mass of 69 kDa for the refolded PHA synt hase. However, light-scattering analysis of a 10-fold concentrated sample i ndicated a molecular mass of 128 kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.