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.).
Cardamone
M
M
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Vic., Australia.
Puri
N K
NK
Sawyer
W H
WH
Capon
R J
RJ
Brandon
M R
MR
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology*; D002942:Circular Dichroism; D013006:Growth Hormone / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D002593:Cetrimonium Compounds; D011994:Recombinant Proteins; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D013006:Growth Hormone; D000077286:Cetrimonium