Cloning and Biochemical Characterization of HIV Integrase
Abstract
Our long range goal is to understand the macromolecular interactions responsible for the integration of HIV DNA into human chromosomes. The primary goal of this project was to develop biochemical assays for the interaction of the HIV-1 integrase protein with its DNA target, the viral LTRs. The gene encoding IN was subcloned from infectious viral DNA and was expressed in both E. coli and in insect cells. The lack of any additional carboxy-terminal processing of IN was demonstrated. Several different solubilization procedures were developed for the purification of integrase. DNA substrates for IN have been constructed; these include synthetic oligonucleotide substrates corresponding to the LTRs and a circular substrate containing the liqated junction of the two LTRs, obtained by polymerase chain amplification from HIV-infected cells. Recombinant protein purified from E. coli is active in a trimming assay in which 2 base pairs are removed from the 3' end of an oligonucleotide substrate. Integration of one labeled substrate into another was also observed. E. coli strains expressing integrase were found to be resistant to infection by single- stranded DNA and RNA bacteriophages.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 1991
- Accession Number
- ADA239634
Entities
People
- Ellen Murphy
Organizations
- Public Health Research Institute