Characterization of a truncated form of recombinant porcine growth hormone generated in vitro during solubilization of inclusion bodies.

Abstract During the development of a novel solubilization procedure (1) for bacterial inclusion bodies (IB′s) using the cationic surfactant cetyltrimethylammonium chloride (CTAC; (CH 3 ) 3 N + C 16 H 33 Cl) significant proportions of an apparently truncated, lower molecular weight (MW) variant form of recombinant pig growth hormone (rPGH) were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis relative to pig pituitary derived GH. The formation of this rPGH-like species, designated P-band, was found to occur in vitro during solubilization of IB′s by CTAC and was dependent on pH and temperature of solubilization, but was not due directly to the use of CTAC, as purified soluble rPGH of the correct MW could not be converted to P-band by exposure to CTAC alone. The bacterial proteolysis suspected as being responsible for the in vitro formation of P-band could not be inhibited by the use of a "cocktail" of defined antiproteolytic agents but was inhibited by pH and temperature, and by solubilization of IB′s in 5% SDS, 6 M gnHCl or 7.5 M urea. Detailed characterization of the structure of P-band by N-terminal amino acid sequencing, electrospray mass spectrometry, radio-receptor binding assay, peptide mapping, and C-terminal peptide sequencing confirmed that P-band was approximately 950 mass units smaller than normal rPGH and lacked eight C-terminal amino acids. A significant finding was that P-band is unable to bind to the pig liver-membrane GH receptor in a competitive radioreceptor assay. Analysis of the relative secondary and tertiary structure of P-band by circular dichroism spectra, intrinsic tryptophan-dependent fluorescence, and average surface hydrophobicity (2) suggested small but measurable changes to the overall structure of P-band relative to normal rPGH. Consequently, our results also suggest that the C-terminal portion of rPGH, including in particular the last eight amino acids, is of major importance in the binding of rPGH to the pig liver membrane GH receptor.