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 6 M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni(2+)-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 10.8 m-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% alpha-helix, 50% beta-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69 kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128 kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.

Genetics and Biochemistry of Polyhydroxyalkanoate Granule Self-assembly: The Key Role of Polyester Synthases

“…Dimerization is significantly enhanced in the presence of substrate or trimeric CoA analogs (3-hydroxybutyryl) 3 -CoA, respectively (Rehm et al 2001, Wodzinska et al 1996 (Figure 2). Six conserved blocks, eight conserved amino acid residues and a conserved a/b-hydrolase fold region were identified based on multiple alignment as well as a conserved domain search of polyester synthase primary structures , Rehm 2003.…”

“…from C. necator, Allochromatioum vinosum, Pseudomonas aeruginosa (PhaC1 and PhaC2) and P. oleovorans (PhaC1) , Mu¨h et al 1999, Qi et al 2000, Rehm et al 2001. The class II polyester synthases were only recently purified and provision of 3-hydroxydecanoyl-CoA as substrate was sufficient for in vitro synthesis of poly(3-hydroxydecanoate) (Qi et al 2000, Rehm et al 2001). …”

Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays

“…In these instances, inclusion bodies were collected by centrifugation and the insoluble proteins were dissolved in 9M urea - 50 mM Tris pH 8.0 buffer. The protein was solubilized in 8M Urea and 5mM TCEP-HCl as reducing agent, was bound to the Ni column, and refolded on-column using a urea gradient [23] (Rehm et. al., 2001).…”

Immunization with the Entamoeba histolytica Surface Metalloprotease EhMSP-1 Protects Hamsters from Amebic Liver Abscess

“…Four recombinant EhMSP-1 fragments comprising the extracellular portion of EhMSP-1 were expressed in Escherichia coli for use as vaccine antigens ( 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) in E. coli strain BL21(DE3) and purified from inclusion bodies by solubilization in accordance with the QIAexpressionist handbook (25) and the work of Rehm et al (26). Contaminating endotoxin was removed from the protein stocks using polymyxin columns (Pierce, Rockford, IL, USA), and residual endotoxin was quantified using a commercial kit (QCL-1000; Lonza, Williamsport, PA, USA).…”