[24] Expression of bovine growth hormone derivatives in Escherichia coli and the use of the derivatives to produce natural sequence growth hormone by cathepsin C cleavage

This chapter discusses the expression of bovine growth hormone (bGH) derivatives in Escherichia coli. The chapter describes the use of the derivatives to produce natural sequence growth hormone by Cathepsin C cleavage. To produce the phenylalanyl form of bGH without an aminoterminal methionine, an efficient enzymatic cleavage method using cathepsin C (diaminopeptidyl peptidase 1, Boehringer-Mannheim) was developed. Cathepsin C is a diaminopeptidase that removes two amino acids, as a unit, from the amino terminal end of the molecule. The enzyme is unreactive with a substrate when the amino terminal amino acid is blocked, Cathepsin C will not cleave on either side of the imino nitrogen of proline, and the enzyme is inactive if lysine or arginine is the amino terminal amino acid of the substrate. The enzyme requires halide ions and a sulfhydryl compound for its activity. The chapter discusses the chemical synthesis and purification of oligonucleotides, enzymatic ligations and the constructions of bGH expression plasmids. The DNA sequence required for the high-level expression of bGH derivatives in Escherichia coli was investigated using temperature-sensitive copy control plasmids as cloning vectors.