Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with β-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-β-galactopyranoside the fusion protein accumulated to a yield of 15–20% of the total cellular protein. After cyanogen bromide deavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isolectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glydne was subsequently amidated by PAM (peptidylglycine-α-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidatlon of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.