Isolation of mutants of human immunodeficiency virus protease based on the toxicity of the enzyme in Escherichia coli.

Baum, Ellen Z.; Bebernitz, Geraldine; Gluzman, Y

doi:10.1073/pnas.87.14.5573

Cited by 49 publications

(38 citation statements)

References 26 publications

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“…Transformants could not be established in the equivalent E. coli strain lacking this plasmid [BL21(DE3)], presumably owing to toxicity of the expressed PR14 protein. Similar observations have been made for expression plasmids encoding HIV-1 protease (Baum et al, 1990). However levels of expression of PR14 in BL21(DE3)pLysS were low, in the order of 50 to 100 ~tg/1 bacterial culture.…”

Section: Expression and Purification Of Htl V-1 Protease (Pr14)supporting

confidence: 53%

Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding

Daenke

Schramm

Bangham

1994

Journal of General Virology

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Human T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expressed in bacteria and purified by gel filtration. A continuous spectrophotometric assay was used to measure the kinetic parameters of substrate hydrolysis by PR14. Several peptide substrates containing HTLV-1 sequences known to be cleaved by PR14 were used. Cleavage analysis showed that the affinity with which PR14 binds these substrates is higher than that previously reported for HTLV-1 Gag peptides. Also, the affinities of peptides containing the sites involved in autocleavage of protease from its precursor are higher than for the peptides containing sites required for structural protein maturation. This suggests that the autocatalysis of protease from its own precursor has priority over other cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases. As the N-and Cterminal regions of retroviral aspartic proteases are known to contribute to stability of the dimer by forming antiparallel fl-strands, short peptides corresponding to these terminal sequences of HTLV-1 protease were tested for their ability to inhibit cleavage of substrates by PRI4. Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, whereas a peptide containing a sequence situated further from the C terminus was less effective. An inhibitor of the protease of human immunodeficiency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR14.

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Section: Expression and Purification Of Htl V-1 Protease (Pr14)supporting

confidence: 53%

Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding

Daenke

Schramm

Bangham

1994

Journal of General Virology

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“…These proteins are hypothesized to cause toxicity by (a) impairing export machinery for secreted proteins [22], (b) by the overexpression of secreted proteins [20,22], (c) by proteolysis [21] and (d) by depletion of tRNA [24]. The mechanism of polyglutamine-mediated cell death is unknown and the polyglutamine constructs produce intracellular proteins with no known enzymatic function.…”

Section: Discussionmentioning

confidence: 99%

“…Numerous recombinant proteins are toxic in E. coli (rat insulin [20]; HIV protease [21]; Streptococcus pneumoniae ma1 and ami loci [22]; cytochrome cp [23] and mutations in tRNA synthetase [24]). These proteins are hypothesized to cause toxicity by (a) impairing export machinery for secreted proteins [22], (b) by the overexpression of secreted proteins [20,22], (c) by proteolysis [21] and (d) by depletion of tRNA [24].…”

Section: Discussionmentioning

confidence: 99%

Toxicity of expanded polyglutamine‐domain proteins in Escherichia coli

et al. 1996

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Five neurodegenerative diseases are caused by proteins with expanded polyglutamine domains. Toxicity of these proteins has been previously identified only in mammals, and no simple model systems are available. In this paper, we demonstrate in E. coli that long polyglutamine domains (59-N residues) as GST-fusion proteins inhibit growth while smaller glutamine (10-35 residues) or polyalanine (61 residues) domains have no effect. Analogously in humans, polyglutamine repeats less than 3540 glutamines produce a normal phenotype, while expansion greater than 40 glutamines is always associated with disease. Expression of polyglutamine proteins in E. coli may help identify the molecular mechanism of pathogenesis of CAG trinucleotide repeat diseases and be a useful screen to identify potential therapeutic compounds.

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“…The currently available detection methods are performed mainly in yeast and bacterial cells or in vitro. The most simple in vivo assays exploit the inherent toxicity of the protease for bacterial cells (2), and more sophisticated strategies have involved the introduction of HIV-1 protease cleavage sites into selectable markers such as ␤-galactosidase (1), tetracycline resistance protein (4), thymidylate synthase (13), galactokinase (24), transcription factors of Saccharomyces cerevisiae (17), and the cI repressor of bacteriophage (21). These assays usually monitor only one or few of the native HIV-1 protease specificity sites.…”

Section: Discussionmentioning

confidence: 99%

Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity

Lindsten

Uhlı́ková

Konvalinka

et al. 2001

Antimicrob Agents Chemother

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The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.The protease encoded by human immunodeficiency virus type 1 (HIV-1) plays an essential role in the retroviral life cycle by processing the viral p55Gag and p160 Gag-Pol polyprotein precursors into structural proteins and enzymes. The activity of the protease is required for conformational rearrangement of the immature virion and production of infectious virus particles, thus providing an attractive target for development of antiviral agents to treat AIDS and related disorders (29). Several potent HIV-1 protease inhibitors are widely used in clinics (7, 10). However, the constant emergence of resistant strains due to the additive effect of multiple amino acid substitutions within and outside the catalytic site motivates the continuous development of new protease inhibitors (26). The availability of reliable and convenient assays for protease activity is, in this context, of great importance.The majority of assays available today are based on trans-or autocatalytic cleavage of reporter proteins in bacteria, in yeasts, or in vitro (1,8,17,24) or on the in vitro hydrolysis of synthetic peptides encompassing the scissile bonds in p55 Gag and p160 14,23). However, none of these assays allows probing of all the native HIV-1 protease specificity sites under physiologic conditions, a situation for which a human cell environment would be required. An important reason for the lack of convenient mammalian cell-based assays is the cytotoxicity observed upon expression of the protease in cells. Thus, while this retroviral aspartic protease possesses unique structural and functional properties that distinguish it from its cellular counterparts (6), several cellular proteins are efficient substrates of the protease. Among those are cytoskeletal proteins such as vimentin, actin, troponin, and tropomyosin (20, 22), microtubule-associated proteins (30), bcl-2 (25), and precursors of NF-B (19) providing a likely explanation for the capacity of the protease to induce apoptosis.We report here on the dev...

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Isolation of mutants of human immunodeficiency virus protease based on the toxicity of the enzyme in Escherichia coli.

Cited by 49 publications

References 26 publications

Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding

Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding

Toxicity of expanded polyglutamine‐domain proteins in Escherichia coli

Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity

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