Nirdosh K. Puri

A spectroscopic and equilibrium binding analysis of cationic detergent-protein interactions using soluble and insoluble recombinant porcine growth hormone.

Overexpression of cloned eukaryote genes in bacteria often leads to the formation of insoluble refractile bodies which require solubilization by harsh denaturants or detergents. We describe the conformational changes associated with the binding of a surfactant, cetyltrimethylammonium chloride (CTAC) to recombinant porcine growth hormone (PGH). The stoichiometry of binding by CTAC to the soluble and insoluble forms of recombinant PGH was also assessed. Optimum CTAC binding and protein solubilisation were obtained at 50 degrees C and at extreme pH. Increased ionic strength and changes in pH towards the isoelectric point of PGH (pH 6) decreased both the binding of CTAC and the efficiency of solubilising PGH from inclusion bodies. The positive charge on the quaternary ammonium head group of CTAC was found to be critical in the binding of CTAC to PGH and for the subsequent solubilisation of inclusion bodies. The binding of CTAC to the soluble form of PGH caused appreciable changes to the tertiary structure of the protein but did not significantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that urea, guanidine hydrochloride and CTAC solubilized recombinant PGH molecules behave differently during in vitro refolding (Puri, N.K., Crivelli, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Brandon, M.R. (1992) Biochem. J. 285, 871-879.).

Production of methionyl-minus ovine growth hormone in Escherichia coli and one-step purification

The overproduction of ovine growth hormone (oGH) in Escherichia coli is described, achieved in part by alteration of the codon usage for nine of the first 15 amino acids (aa) of the mature hormone. Recombinant oGH (re-oGH), representing 12% of the total cellular protein, was isolated from inclusion bodies by solubilisation using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The hormone was refolded and subsequently purified to greater than 95% homogeneity in a single step using preparative reverse phase high performance liquid chromatography. The aa sequence analysis revealed that the N-terminus of the E. coli-derived polypeptide was identical to that of pituitary-derived oGH, and re-oGH displayed potent somatotropic activity in vivo.

A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding

Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IBs) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urca or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2°) structure of the urea‐ and GnHCl‐solubilised rPGH showed the absence of α‐helical content with the majority of the molecule existing in a ‘random coil’ structure. In contrast, the CTAC‐solubilised rPGH displayed significant starting 2° structure (10–15% α helix; 30–40% β structure). The three rPGH preparations were refolded in vitro against weak urea, GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IBs and only 20% for urea or GnHCl solubilised IBs. We conclude that the method of solubilisation of IBs and the resultant difference in the starting 2° structure of rPGH, particularly α‐helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.

Monoclonal antibodies to sheep MHC class II molecules recognize all HLA-D or subsets of HLA-D region products.

Six murine monoclonal antibodies raised against sheep MHC class II molecules were analyzed for reactivity with HLA-D subregion products. All the antibodies reacted with human peripheral blood lymphocytes, monocytes, and B-lymphoblastoid cell lines homozygous for various HLA-DR specificities, suggesting that the antibodies recognized nonpolymorphic determinants on HLA class II molecules. SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analyses of molecules immunoprecipitated from 35S-methionine-labeled, DR-homozygous B-lymphoblastoid cell lines showed that the monoclonal antibodies precipitated typical class II molecules (Mr 32-34K and 25-29K). From comparison with antibodies of known HLA-D subregion specificity, two of the sheep antibodies appeared to react with the products of single HLA-D subregions, while another showed balanced reactivity with all HLA-D molecules. Antibody SBU.II 38-27 reacted exclusively with HLA-DQ molecules, antibody SBU.II 28-1 with HLA-DP molecules, and antibody SBU.II 49-1 with HLA-DR, -DQ, and -DP molecules. However, analysis of immunoprecipitates from surface-iodinated WT-49 cells (DR3 homozygous) using SBU.II 28-1 and the DP-specific monoclonal antibody B7/21, suggested that the two antibodies immunoprecipitated different alpha polypeptides. The two antibodies SBU.II 38-27 and 28-1 appear to be at least as specific as existing reagents, if not more so. As such, they are of value in their potential contribution to our understanding of the molecular characteristics and ultimately the functions of the HLA-DQ and -DP subregion products, as well as the identification/characterization of HLA-D equivalents in other species.