Peter Hobart

Crystallizable HIV-1 protease derived from expression of the viral pol gene in Escherichia coli

A plasmid vector was used to express the HIV-1 pol open reading frame under the regulation of the bacterial trp promoter in Escherichia coli. This expression system has been used as a source of recombinant viral protease. The self-processed active enzyme was recovered from a soluble fraction of a bacterial cell lysate and purified by a procedure involving four steps of chromatography. The protocol yielded 0.3 mg of protease for each liter of bacterial culture. The protease formed tetragonal bipyramidal crystals which have been used in high-resolution X-ray diffraction studies.

Persistent cytoplasmic location of a DNA polymerase β in sea urchins during development

A subcellular localization study of a low molecular weight DNA polymerase beta indicates that this enzyme, as well as a high molecular weight DNA polymerase alpha, is found in large quantities in the cytoplasm of Strongylocentrotus purpuratus eggs. The two enzyme activities are distinguished by DEAE-sievorptive chromatography and by their differential activities with activated DNA and oligo(dT)10 . poly(dA)200 primer-templates. Using an enucleation procedure, it is concluded that an extremely low proportion if any, of both polymerases is present in the egg nucleus. At blastula stage, a period of rapid cell proliferation, similar studies of DNA polymerase subcellular localization using two different methods of nuclear isolation indicate that the DNA polymerase beta remains largely cytoplasmic while the alpha enzyme is found to be predominantly nuclear. Since the results for the alpha enzyme agree with previous reports (Loeb, L.A. (1969) J. Biol. Chem. 244, 1672) and since one method of nuclear isolation, using hypotonic solutions, enables us to recover both DNA polymerase alpha and beta activities in isolated mouse L-cell nuclei, the enzyme quantitation of isolated sea urchin nuclei is considered accurate. Thus, although there is a translocation of the polymerase alpha from a cytoplasmic to nuclear site during early embryonic development, such a massive relocalization of the polymerase beta does not occur.

Three-dimensional Structure and Evolution of HIV-1 Protease

HIV-1 proteinase processes its virally encoded polyproteins into mature structural proteins and enzymes that are essential for viral propagation. As a consequence the proteinase is an attractive target for prospective antiviral agents for the treatment of AIDS, and knowledge of its tertiary structure an important step in drug design. Following the observation (Toh et al. 1985) that retroviral proteinases shared a highly conserved sequence Asp-Thr/Ser-Gly with the pepsins, it has been hypothesised (Pearl and Taylor, 1987; Blundell et al. 1988) on the basis of sequence analysis and modelling studies that these enzymes exist as dimers closely similar in three-dimensional structure to the ancestral dimeric proteinase suggested for the aspartic proteinases (Tang et al. 1978). This has now been confirmed, first by X-ray analysis of a synthetic HIV-1 proteinase in the laboratory of Wlodawer (Weber et al. 1989) and then for a recombinant enzyme in our own laboratories (Lapatto et al. 1989). These X-ray structure analyses indicated that the overall fold of the HIV-1 proteinase closely resembled that of the RSV-proteinase (Miller et al., 1989). The Asp-Thr-Gly sequences adopt a conformation closely similar to that of the pepsin-like aspartic proteinases but organised symmetrically in the dimer about the crystallographic 2-fold axis. However, the N- and C-termini together form an intermolecular four-stranded sheet, which is central to the stability of the dimer, in contrast to the inter-subunit sheet of the pepsins, which has six antiparallel strands arranged around the pseudo dyad.