ABSTRACT
Genes were constructed to encode single-chain tethered human immunodeficiency virus HIV-1/HIV-1 and HIV-2/HIV-2 homodimeric proteinases and two HIV-1/HIV-2 heterodimers which differed in the nature of the interface strands. All four constructs under the control of a heat-inducible promoter were expressed in E. coli and the resultant proteinases were purified therefrom. Kinetic parameters (Km, kcat and kcat/Km) were derived for the interaction of the tethered homo and heterodimeric proteinases with two distinct substrates at a variety of pH values. All four enzymes were comparably active toward one substrate. With the second substrate at pH 4.7, the kcat/Km value was best for the HIV-1/1 tethered homodimer, 15-fold lower for the two heterodimeric proteinases, and was reduced by an additional 6-fold for the HIV-2/2 homodimer. From the Ki values determined for the interactions of the four tethered dimer proteinases with a systematic series of synthetic inhibitors, a parallel trend was observed. Whereas several inhibitors were equipotent against all four enzymes, two were discriminatory in that they inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV -2/2 tethered homodimer (or its untethered wild-type counterpart from HIV-2). The significance of these findings for active site interaction with HIV-proteinases is considered.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , HIV-1/enzymology , HIV-2/enzymology , Amino Acid Sequence , Aspartic Acid Endopeptidases/biosynthesis , Cloning, Molecular , Escherichia coli , HIV Protease/biosynthesis , HIV Protease Inhibitors/metabolism , Kinetics , Macromolecular Substances , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Protein Multimerization , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Structure-Activity Relationship , Substrate SpecificitySubject(s)
Eimeria tenella/genetics , Genes, Protozoan , NADP Transhydrogenases/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , DNA, Protozoan/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Mitochondria/enzymology , Molecular Sequence Data , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
The transcriptional activator protein (Tax) from human T-cell leukemia virus type 1 was expressed in yeast using several different promoters in several strains: In all instances, expression of Tax resulted in very strong aggregation of the yeast cells. This phenotype appears to be identical by all criteria tested to the flocculation phenotype of the dominant mutation flo 1. Of most significance, mutations in Tax that affect transactivation of the IL-2R alpha regulatory sequences, but retain their ability to activate the viral long terminal repeat also fail to yield the aggregation phenotype. Based on these findings, expression of Tax in yeast may prove to be a simple primordial system for examining the regulatory mechanisms and cellular functions involved in regulation of gene expression.
Subject(s)
Gene Products, tax/genetics , Human T-lymphotropic virus 1/genetics , Saccharomyces cerevisiae/genetics , Transcriptional Activation , Gene Products, tax/physiology , Human T-lymphotropic virus 1/metabolism , Immunoblotting , Mutation , Phenotype , Promoter Regions, Genetic , Saccharomyces cerevisiae/cytologyABSTRACT
Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated. Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis. Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix. Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein. Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons. This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons.
Subject(s)
Gene Products, rev/isolation & purification , HIV-1/genetics , Chromatography, Gel , Circular Dichroism , Cloning, Molecular , Escherichia coli/genetics , Gene Products, rev/genetics , Gene Products, rev/metabolism , Genes, Synthetic , HIV-1/metabolism , Kinetics , Plasmids , Protein Binding , Protein Conformation , Recombinant Proteins/isolation & purification , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , rev Gene Products, Human Immunodeficiency VirusABSTRACT
A genetic approach was used to facilitate purification of human immunodeficiency virus (HIV) rev protein. A recombinant protein containing a stretch of six histidine residues at the amino terminus was engineered and overexpressed in Escherichia coli. Purification of greater than 95% was achieved in a single step using an immobilized metal ion chromatography with a resin that has selectivity for proteins with neighboring histidine residues. We show that the modified protein is both properly modified and biologically active.
