Large scale purification and refolding of HIV-1 protease fromEscherichia coli inclusion bodies

Hui, John O.; Tomasselli, Alfredo G.; Reardon, Ilene M.; Lull, June M.; Brunner, David P.; Tomich, Che-Shen C.; Heinrikson, Robert L.

doi:10.1007/bf01028194

Cited by 58 publications

(45 citation statements)

References 14 publications

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“…The alanine mutation at position 82 was introduced by using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). The protocol used for protein overexpression and purification has been described previously (21,43,48).…”

Section: Methodsmentioning

confidence: 99%

Viability of a Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variant: Structural Insights for Better Antiviral Therapy

Prabu-Jeyabalan

Nalivaika

King

et al. 2003

J Virol

102

121

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Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.

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Section: Methodsmentioning

confidence: 99%

Viability of a Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variant: Structural Insights for Better Antiviral Therapy

Prabu-Jeyabalan

Nalivaika

King

et al. 2003

J Virol

102

121

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“…Briefly, the HIV protease gene was cloned into plasmid pXC-35 (American Type Culture Collection, Manassas, Va.) (7), which was transfected into E. coli TAP106. Transfected cells were grown in a 12-liter fermentor and, following protein expression, lysed to release the inclusion bodies containing the protease (20). Inclusion bodies were isolated by centrifugation, and the pellet was dissolved in 50% acetic acid to extract protease.…”

Section: Methodsmentioning

confidence: 99%

Role of Invariant Thr80 in Human Immunodeficiency Virus Type 1 Protease Structure, Function, and Viral Infectivity

Foulkes

Prabu-Jeyabalan

Cooper

et al. 2006

J Virol

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Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.

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“…1). The protease was expressed as the full-length enzyme, residues 1-99; about 5% of the enzyme consisted of chains 100 residues in length with Met at position 1 (Tomasselli et al, 1990c;Hui et al, 1993). Solvents for protein sequencing, amino acid analysis, HPLC, SDS-PAGE, and other reagents used in the present studies, were of the highest grade commercially available.…”

Section: Methodsmentioning

confidence: 99%

Human immunodeficiency virus type‐1 reverse transcriptase and ribonuclease h as substrates of the viral protease

Tomasselli¹,

Sarcich²,

Barrett³

et al. 1993

Protein Science

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A study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV-1) to digestion by the HIV-1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440-Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (~6 6 1~5 1 ) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227-14232). The COOH-terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr,,,-Leu,,, and Leu,,,. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly,,,-Ala,,, and at Phe440-Tyr441, and only much more slowly at residues 483,494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483,494, and 532 are relatively inaccessible in comparison to Gly,,, and Phe,,. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a plS segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.

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Large scale purification and refolding of HIV-1 protease fromEscherichia coli inclusion bodies

Cited by 58 publications

References 14 publications

Viability of a Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variant: Structural Insights for Better Antiviral Therapy

Viability of a Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variant: Structural Insights for Better Antiviral Therapy

Role of Invariant Thr80 in Human Immunodeficiency Virus Type 1 Protease Structure, Function, and Viral Infectivity

Human immunodeficiency virus type‐1 reverse transcriptase and ribonuclease h as substrates of the viral protease

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