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-I 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, Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQVF32-OH (I; GRF32) and Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQ30-OH (IA; GRF30), conform well to the specificity of the HIV-I protease for residues in the P1' to P4' positions of its peptide substrates. A variety of amino acids were tried in the N-terminal extension (positions P4-P1) to fit the protease substrate specificity for the 8 amino acids in positions P4-P4'. 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-1-Tyr1 bond (... RQVF-decreases-YIDA ...) to release GRF32. However, when several soluble fusion proteins linked to GRF32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF32 met with variable, and only limited, success. By random mutagenesis in a propeptide segment, [MGQSVAQVF]-decreases-GRF30, (II) was identified as a construct that showed reasonably high-level expression in E. coli and was effectively processed by the HIV-I protease. A yield of 5 mg of pure GRF30 was obtained/litre of culture medium after a single HPLC purification step.
Tomasselli
A G
AG
Biochemistry, Pharmacia & Upjohn, Kalamazoo, MI 49007, USA.
Mildner
A A
AA
Paddock
D J
DJ
Wheeler
J S
JS
Kubiak
T M
TM
Martin
R A
RA
Moseley
W M
WM
Mott
J E
JE
White
M C
MC
Leone
J W
JW
Heinrikson
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D013007:Growth Hormone-Releasing Hormone / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013007:Growth Hormone-Releasing Hormone; D016333:HIV Protease