Use of the HIV-1 protease for excision of growth-hormone-releasing factor from synthetic and recombinant peptide precursors.
Biotechnol Appl Biochem
; 26(1): 39-49, 1997 08.
Article
en En
| MEDLINE
| ID: mdl-9262002
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fragmentos de Péptidos
/
Precursores de Proteínas
/
Proteasa del VIH
/
Hormona Liberadora de Hormona del Crecimiento
/
VIH-1
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Biotechnol Appl Biochem
Asunto de la revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Año:
1997
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos