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Expression of an autoprocessing CAT-HIV-1 proteinase fusion protein: purification to homogeneity of the release 99 residue proteinase.




 

Biochem Biophys Res Commun. 1991 Mar 29;175(3):784-94. Unique Identifier

The 99 residue human immunodeficiency virus type 1 proteinase has been expressed in Escherichia coli as part of an autocleaving fusion protein. Expression of the fusion protein is toxic to the host cells, however yields of the released proteinase have been improved by optimising induction nad harvest times to increase culture biomass, and decrease degradation of the proteinase. Soluble proteinase was extracted from these cells by a simple and highly efficient three step process. N-terminal sequence analysis confirms that the enzyme preparation is highly pure and correctly autoprocessed. The proteinase cleaves peptide substrate IGCTLNFPISPIETV between F and P at pH 6.0 with a Km of 310 microM and a Kcat of 14s-1. The enzyme is sensitive to its ionic environment, showing stimulation of activity at high salt concentrations, and shows a pH optimising 5.5.

Amino Acid Sequence Androgen-Binding Proteins Base Sequence Chloramphenicol Acetyltransferase/*GENETICS Chromatography, Gel Chromatography, High Pressure Liquid Chromatography, Ion Exchange Cloning, Molecular Electrophoresis, Polyacrylamide Gel Escherichia coli/GENETICS Gene Expression/DRUG EFFECTS HIV Protease/*GENETICS/ISOLATION & PURIF/METABOLISM HIV-1/ENZYMOLOGY/*GENETICS Isopropyl Thiogalactoside/PHARMACOLOGY Kinetics Molecular Sequence Data Molecular Weight Oligonucleotide Probes Plasmids Recombinant Fusion Proteins/ISOLATION & PURIF Recombinant Proteins/ISOLATION & PURIF/METABOLISM Restriction Mapping JOURNAL ARTICLE



 




Information in this article was accurate in August 30, 1991. The state of the art may have changed since the publication date. This material is designed to support, not replace, the relationship that exists between you and your doctor. Always discuss treatment options with a doctor who specializes in treating HIV.