Use of the HIV-1 protease for excision of growth-hormone-releasing factor from synthetic and recombinant peptide precursors

An autolysis-resistant mutant of the HIV-1 protease was employed for removal of metabolically stabilized and highly bioactive analogues of bovine growth-hormone-releasing factor (bGRF) from their larger either synthetic or recombinant precursors. The N-terminal four amino acids in two selected model GRF analogues, Y 1 IDAIFTSSYRKVLAQLSARKLLQ-DILSRQVF 32 -OH (I; GRF 32 ) and Y'IDAIFTSSYRKV-LAQLSARKLLQDILSRQ 30 -OH (IA; GRF 30 ), conform well to the specificity of the HIV-1 protease for residues in the P 1 ' to P 4 ' positions of its peptide substrates. A variety of amino acids were tried in the N-terminal extension (positions P 4 -P 1 ) to fit the protease substrate specificity for the 8 amino acids in positions P 4 -P 4 ' A synthetic precursor of I, extended N-terminally with RQVF-, a sequence representing the four C-terminal residues in I, was effectively cleaved by the protease at the Phe '-Tyr' bond (...RQVF-↓-YIDA...) to release GRF 32- However, when several soluble fusion proteins linked to GRF 32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF 32 met with variable, and only limited, success. By random mutagenesis in a propeptide segment, [MGQSVAQVF]-↓-GRF 30 , (II) was identified as a construct that showed reasonably high-level expression in E. coli and was effectively processed by the HIV-1 protease. A yield of 5 mg of pure GRF 30 was obtained/litre of culture medium after a single HPLC purification step.