Site directed mutagenesis, autoprocessing and inhibitor studies on the retroviral protease of the human immunodeficiency virus type-1

Garner, Joanne Clare (1999) Site directed mutagenesis, autoprocessing and inhibitor studies on the retroviral protease of the human immunodeficiency virus type-1. University of Southampton, Doctoral Thesis.

Abstract

A synthetic gene encoding the human immunodeficiency type-1 protease, expressed in E. coli, was used to investigate several types of mutations produced by the PCR method of mutagenesis and also to explore the autoprocessing of the HIV-1 protease from its precursor protein. The gene was originally expressed from the vector pT7-5 in K38 pGP1-2, as a cysteine-67-alanine; cysteine-95-alanine double mutant (C67A and C95A) to avoid potential problems with cysteine oxidation. These mutations were shown to have no significant effect on the enzyme activity as judged by their ability to cleave the chromogenic substrate, KARV's substrate. In contrast, proteins with mutations at aspartate-25 (D25A and D25E) and alanine-28 (A28C) were totally devoid of activity and were isolated as precursor proteins with their N-termini unprocessed.

The importance of the N-terminal pre-sequence in relationship to autoprocessing from the precursor protein was investigated by mutating several amino-acids adjacent to the scissile bond (SFNF/PQIT). Subtle differences in amino-acid presequence, (M)GTVSFNF → (M)ΔTVSFNF, led to the complete inability of the precursor to autoprocess. Several other mutations in this region ((M)ATVSFNF, (M)PTVSFNF, (M)GΔVSFNF, (M)GTLSFNF, (M)QGTVSFNF, (M)RQGTVSFNF, (M)DRQGTVSFNF) were generated to study the details of autoprocessing further.

The approved anti-AIDS drugs, Ritonavir and Saquinavir and a microbial alkaline proteinase inhibitor, α-MAPI 1, were studied with the recombinant HIV-1 protein to determine their efficacy.

This record has no associated files available for download.

Contributors

Download statistics