HIV-I protease. Cloning, expression, and purification.
A new method for obtaining HIV-I protease was suggested. Fusion proteins composed of the N-terminal fragment of human gamma-interferon and HIV-I protease connected with (Asp)4Lys (protein I) or Asp-Pro (protein II) linkers were expressed in Escherichia coli cells. The fusion proteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein. Protein I was cleaved by enterokinase. The solubility of protein I was increased by treating with Na-sulfite/Na-tetrathionate under denaturing conditions. Optimal conditions for efficient acidic hydrolysis of protein II at Asp-Pro bond were found. The hydrolysis products were separated by reversed-phase FPLC. The amount of tryptophan and cysteine residues in the enzyme obtained was estimated. The activity of HIV-I protease was determined using the chromogenic peptide. AlaArgVal NleNphGluAlaNleNH2 and a high-mol-wt substrate consisting of beta-galactosidase and a fragment of gag proteins, including p17-p24 processing site.
Dergousova
N I
NI
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. nd@enzyme.siobc.ras.ru
Amerik AYu
Volynskaya
A M
AM
Rumsh
L D
LD
D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000378:metabolism; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry; D008970:Molecular Weight
D007371:Interferon-gamma; D004765:Enteropeptidase; D016333:HIV Protease