Abstract
The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E. coli. A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Base Sequence
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Blotting, Western
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Cloning, Molecular*
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DNA / chemical synthesis*
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DNA / genetics
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Endopeptidases / genetics*
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Endopeptidases / metabolism
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Escherichia coli / enzymology
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Escherichia coli / genetics*
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Gene Expression Regulation*
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Gene Products, gag
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HIV Protease
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Kinetics
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Molecular Sequence Data
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Nucleic Acid Hybridization
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Peptide Fragments / metabolism
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Plasmids
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Protein Precursors / metabolism
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Retroviridae Proteins / metabolism
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Substrate Specificity
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Transformation, Bacterial
Substances
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Gene Products, gag
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Peptide Fragments
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Protein Precursors
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Retroviridae Proteins
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DNA
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Endopeptidases
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HIV Protease
Associated data
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GENBANK/M24549
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GENBANK/M24550
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GENBANK/M24551
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GENBANK/M24552
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GENBANK/M24553