Gary C. Harbour

Reversed-phase high-performance liquid chromatography peptide mapping of bovine somatotropin☆

Reversed-phase high-performance liquid chromatography peptide mapping techniques have been used to examine the primary structure of bovine somatotropin (bSt) isolated from Escherichia coli modified by recombinant DNA techniques (rbSt) and from bovine pituitary (pbSt). Peptide fragments arising from tryptic digestion of bSt were separated on Baker wide pore C4 or C8 columns with linear gradients (acetonitrile-water with 0.1% trifluoroacetic acid). Major peaks eluted in a consistent order, but significant day-to-day variation in retention times was observed. Isolated peptide fragments were analyzed via acid hydrolysis followed by formation and separation of the phenylthiocarbamyl amino acids. These correspond to expected tryptic fragments.

N-ε-Acetylation Can Occur at Lysine Residues 157, 167, 171, And 180 of Recombinant Bovine Somatotropin

Publisher Summary It was found that a significant fraction of the p 7 rbSt arises from acetylation of certain lysine residues. This chapter discusses the isolation and structural elucidation of the acetylated pI 7 bands in rbSt. Acetylation of lysine is a well known post-translational modification mediated by an acetyltransferase enzyme using acetyl-CoA as a substrate. It has been demonstrated with the histones, where it is believed to regulate the binding of these very basic proteins to the negatively charged ribose backbone of the nucleic acids. The acetylation of lysines on rbSt is a post-translational modification carried out by the host E. coli organism. Acetylation of lysines makes them unrecognizable to trypsin and in each case result in the removal of one positive charge in the parent protein, thus lowering the pi. The data indicate that at least four of the pi 7 species are produced by acetylation of lysines 157, 167, 171, and 180. Another species is formed by the base catalyzed rearrangement of asparagine 99 to isoaspartic acid.