A SPECTROSCOPIC AND EQUILIBRIUM BINDING ANALYSIS OF CATIONIC DETERGENT-PROTEIN INTERACTIONS USING SOLUBLE AND INSOLUBLE RECOMBINANT PORCINEGROWTH-HORMONE

Overexpression of cloned eukaryote genes in bacteria often leads to th e formation of insoluble refractile bodies which require solubilizatio n by harsh denaturants or detergents. We describe the conformational c hanges associated with the binding of a surfactant, cetyltrimethylammo nium 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 s olubilisation were obtained at 50 degrees C and at extreme pH. Increas ed ionic strength and changes in pH towards the isoelectric point of P GH (pH 6) decreased both the binding of CTAC and the efficiency of sol ubilising PGH from inclusion bodies. The positive charge on the quater nary ammonium head group of CTAC was found to be critical in the bindi ng of CTAC to PGK and for the subsequent solubilisation of inclusion b odies. The binding of CTAC to the soluble form of PGH caused appreciab le changes to the tertiary structure of the protein but did not signif icantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that ur ea, guanidine hydrochloride and CTAC solubilized recombinant PGH molec ules behave differently during in vitro refolding (Purl, N.K., Crivell i, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Bran don, M.R. (1992) Biochem. J. 285, 871-879.).