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

Abstract

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.

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“…This study clearly defined that the PHA synthases possessed all the features required for self-organization into spherical particles. This was further supported by establishment of in vitro PHA synthesis using purified PHA synthases from other microorganisms, such as for example from Cupriavidus necator, Allochromatioum vinosum, Pseudomonas aeruginosa (PhaC1 and PhaC2) and P. oleovorans (PhaC1) (114)(115)(116). The class II PHA synthases were only recently purified and provision of 3-hydroxydecanoyl-CoA as a substrate was sufficient for in vitro synthesis of poly(3-hydroxydecanoate) (115,116).…”

Acyltransferases in Bacteria

“…The 3D structure of PHA synthases has not been elucidated yet, but it has been observed that the enzyme is a homodimer in its active form and that its dimerization is induced by the presence of substrate (258,259). PHA synthases catalyze the polymerization at the surface of the growing PHA granule (260,261).…”

Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

“…Reduced temperatures (typically 30°C) are often used to prevent inclusion body formation, and addition of a mild nonionic detergent such as 6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside (Hecameg) is essential to prevent agglomeration of the enzyme during and after purification (3). Some attempts have also been made to resolubilize inclusion bodies formed by the type II synthases from Pseudomonas oleovorans using S-Sepharose (8) and from Pseudomonas putida by denaturing with 6 M guanidine hydrochloride, followed by refolding (9).…”