Cloning, expression and purification of a recombinant poly‐histidine‐linked HIV‐1 protease

Leuthardt, Andreas; Roesel, Johannes

doi:10.1016/0014-5793(93)81807-c

Cited by 13 publications

(10 citation statements)

References 26 publications

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“…Recombinant HIV ‐ 1 ‐ PR , expressed in Escherichia coli (Bachem UK, Ltd., catalog H‐9040)26, 27 contained five mutations to restrict autoproteolysis (Q7K, L33I, L36I) and to restrict cysteine thiol oxidation (C67A and C95A). The enzyme was stored (at −70°C) as solution with concentration 0.1 μg/μL in dilute HCl (pH = 1.6).…”

Section: Methodsmentioning

confidence: 99%

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A folding inhibitor of the HIV‐1 protease

Broglia¹,

Provasi²,

Ottolina³

et al. 2005

Proteins

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Being the HIV-1 Protease (HIV-1-PR) an essential enzyme in the viral life cycle, its inhibition can control AIDS. The folding of single domain proteins, like each of the monomers forming the HIV-1-PR homodimer, is controlled by local elementary structures (LES, folding units stabilized by strongly interacting, highly conserved, as a rule hydrophobic, amino acids). These LES have evolved over myriad of generations to recognize and strongly attract each other, so as to make the protein fold fast and be stable in its native conformation. Consequently, peptides displaying a sequence identical to those segments of the monomers associated with LES are expected to act as competitive inhibitors and thus destabilize the native structure of the enzyme. These inhibitors are unlikely to lead to escape mutants as they bind to the protease monomers through highly conserved amino acids which play an essential role in the folding process. The properties of one of the most promising inhibitors of the folding of the HIV-1-PR monomers found among these peptides is demonstrated with the help of spectrophotometric assays and CD spectroscopy.2

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Section: Methodsmentioning

confidence: 99%

“…The assay buffer was prepared, following the method by Leuthardt and Roesel,27 by adding 0.8 m M NaCl, 1 m M ethylenediaminetetraacetic acid (EDTA) and 1 m M dithiothreitol to a 20 m M phosphate buffer (pH 6).…”

Section: Methodsmentioning

confidence: 99%

A folding inhibitor of the HIV‐1 protease

Broglia¹,

Provasi²,

Ottolina³

et al. 2005

Proteins

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“…In order to produce large amounts of HIV-1Pr, different strategies were investigated: i) it was produced by autocatalytic processing of a larger precursor [4,5]; ii) it was fused to a variety of proteins, e.g., β-lactamase [6], glutathione S-transferase (GST), maltose-binding protein [7], N-terminal portion of γ-interferon [8], or as His-tagged recombinant protein [9]; iii) codon usage, A+T richness at the 5' end of the coding region, and the promoter were optimized [10,11]; and iv) it was recovered by refolding of E. coli inclusion bodies [12,13]. Owing to the cytotoxicity (and low solubility) of this protease, it is difficult to obtain it in large quantities: in most cases the expression level was low and the recombinant HIV-1Pr could be detected only by immunoblotting (see Additional file 1, Table S1).…”

Section: Introductionmentioning

confidence: 99%

Optimizing HIV-1 protease production in Escherichia coli as fusion protein

2011

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BackgroundHuman immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations.ResultsA recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis) and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1Pr per liter.ConclusionsBy using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.

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“…For some purposes this difficulty can be overcome by using an active-site mutant of PR. In addition, retroviral PR domains are extremely insoluble (6,10,21). Expression of an HIV-1 Gag protein with an inactivated PR domain at its C terminus (creating a molecule equivalent to RSV Gag) resulted in a reduction of extracellular VLP production by 5-to 10-fold compared with wild-type Gag, and the particles produced had a lower density and were irregular in shape (19).…”

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confidence: 99%

PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells

Johnson

Scobie

Vogt

2001

J Virol

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While baculovirus expression of Gag proteins from numerous retroviruses has led reliably to production of virus-like particles (VLPs), we observed that expression of Rous sarcoma virus Gag failed to produce VLPs. Transmission and scanning electron microscopy analysis revealed that the Gag protein reached the plasma membrane but was unable to correctly form particles. Addition of a myristylation signal had no effect on the budding defect, but deletion of the PR domain of Gag restored normal budding. The resulting VLPs were morphologically distinct from human immunodeficiency virus type 1 VLPs expressed in parallel.

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Cloning, expression and purification of a recombinant poly‐histidine‐linked HIV‐1 protease

Cited by 13 publications

References 26 publications

A folding inhibitor of the HIV‐1 protease

A folding inhibitor of the HIV‐1 protease

Optimizing HIV-1 protease production in Escherichia coli as fusion protein

PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells

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