Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies

Maseko, Sibusiso B.; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E. M.; Lin, Johnson; Kruger, Hendrik G.

doi:10.1016/j.pep.2016.02.013

Cited by 22 publications

(14 citation statements)

References 32 publications

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“…However, our group recently reported 100 and 101 amino acid mutant variants of C‐SA protease that demonstrated little difference in catalytic activity with respect to the natural substrate . The first mutant has one extra amino acid (I36T↑T) and the second mutant has two additional amino acids (E35D↑G↑S) in each monomer . Limited data from another study, reported by our group demonstrated that FDA approved protease inhibitors exhibit reduced activities against subtype C because of residue polymorphism.…”

Section: Introductionmentioning

confidence: 89%

“…HIV‐1 protease is generally a homodimer with two identical monomers (chain A and chain B) each consisting of 99 amino acids . However, our group recently reported 100 and 101 amino acid mutant variants of C‐SA protease that demonstrated little difference in catalytic activity with respect to the natural substrate . The first mutant has one extra amino acid (I36T↑T) and the second mutant has two additional amino acids (E35D↑G↑S) in each monomer .…”

Section: Introductionmentioning

confidence: 99%

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Exploring the flap dynamics of the South African HIV subtype C protease in presence of FDA‐approved inhibitors: MD study

et al. 2018

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HIV-1 protease (HIV PR) is considered as one of the most attractive targets for the treatment of HIV and the impact of flap dynamics of HIV PR on the binding affinities of protease inhibitors (PIs) is a crucial ongoing research field. Recently, our research group evaluated the binding affinities of different FDA approved PIs against the South African HIV-1 subtype C (C-SA) protease (PR). The CSA-HIV PR displayed weaker binding affinity for most of the clinical PIs compared to HIV-1 B subtype for West and Central Europe, the Americas. In the current work, the flap dynamics of four different systems of HIV-1 C-SA PR complexed to FDA approved second generation PIs and its impact on binding was explored over the molecular dynamic trajectories. It was observed that the interactions of the selected drugs with the binding site residues of the protease may not be the major contributor for affinity towards PIs. Various post-MD analyses were performed, also entropic contributions, solvation free energies and hydrophobic core formation interactions were studied to assess how the flap dynamics of C-SA PR which is affected by such factors. From these contributions, large van der Waals interactions and low solvation free energies were found to be major factors for the higher activity of ATV against C-SA HIV PR. Furthermore, a comparatively stable hydrophobic core may be responsible for higher stability of the PR flaps of the ATV complex. The outcome of this study provides significant guidance to how the flap dynamics of C-SA PR is affected by various factors as a result of the binding affinity of various protease inhibitors. It will also assist with the design of potent inhibitors against C-SA HIV PR that apart from binding in the active site of PR can interacts with the flaps to prevent opening of the flaps resulting in inactivation of the protease.

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Section: Introductionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Exploring the flap dynamics of the South African HIV subtype C protease in presence of FDA‐approved inhibitors: MD study

et al. 2018

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“…The positive clones were incubated in 10 mL of Luria-Bertani (LB) medium (1% bacto tryptone, 0.5% yeast extract, 1% NaCl) containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol (Merck, Germany) overnight at 37 °C at 180 revs per minute (rpm). One millilitre of an overnight culture was used to inoculate 100 mL of LB broth using the above method (Maseko et al 2016;Volontè et al 2011). The expression of human proinsulin was induced by the addition of 0.1 to 1 mM Isopropyl β-d thiogalactoside (IPTG) at an early exponential phase (OD 600 0.4-0.7).…”

Section: The Expression and Isolation Of The Proteinmentioning

confidence: 99%

“…The over-expressed protein was recovered following to a similar method adapted from Maseko et al (2016). After overnight induction at 16 °C, the cultures were transferred to 50 mL Cell Star ® centrifuge tubes (Greiner Bio-One, Austria) and spun down at 8000 rpm for 15 min at 4 °C.…”

Section: The Expression and Isolation Of The Proteinmentioning

confidence: 99%

A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)

et al. 2020

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Insulin has captured researchers' attention worldwide. There is a rapid global rise in the number of diabetic patients, which increases the demand for insulin. Current methods of insulin production are expensive and time-consuming. A PCR-based strategy was employed for the cloning and verification of human insulin. The human insulin protein was then overexpressed in E. coli on a laboratory scale. Thereafter, optimisation of human insulin expression was conducted. The yield of human insulin produced was approximately 520.92 (mg/L), located in the intracellular fraction. Human insulin was detected using the MALDI-TOF-MS and LC-MS methods. The crude biosynthesised protein sequence was verified using protein sequencing, which had a 100% similarity to the human insulin sequence. The biological activity of human insulin was tested in vitro using a MTT assay, which revealed that the crude biosynthesised human insulin displayed a similar degree of efficacy to the standard human insulin. This study eliminated the use of affinity tags since an untagged pET21b expression vector was employed. Tedious protein renaturation, inclusion body recovery steps, and the expensive enzymatic cleavage of the C-peptide of insulin were eliminated, thereby making this method of biosynthesising human insulin a novel and more efficient method.

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“…Efficient treatments against viruses are hindered by the advancement of drug resistance and non-specific targeting, particularly those associated with influenza [18] and HIV [19,20]. Indeed, the main challenges to combat such epidemic viral infections are that for several of them (especially SARS-CoV infections) there are no effective antiviral drugs or specific treatments accessible, thus designing potent drugs to combat these diseases needs to be pursued urgently.…”

Section: Introductionmentioning

confidence: 99%

Nanomaterials and Nanotechnology-Associated Innovations against Viral Infections with a Focus on Coronaviruses

et al. 2020

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Viral infections have recently emerged not only as a health threat to people but rapidly became the cause of universal fatality on a large scale. Nanomaterials comprising functionalized nanoparticles (NPs) and quantum dots and nanotechnology-associated innovative detection methods, vaccine design, and nanodrug production have shown immense promise for interfacing with pathogenic viruses and restricting their entrance into cells. These viruses have been scrutinized using rapid diagnostic detection and therapeutic interventional options against the caused infections including vaccine development for prevention and control. Coronaviruses, namely SARS-CoV, MERS-CoV, and SARS-CoV-2, have endangered human life, and the COVID-19 (caused by SARS-CoV-2) outbreak has become a perilous challenge to public health globally with huge accompanying morbidity rates. Thus, it is imperative to expedite the drug and vaccine development efforts that would help mitigate this pandemic. In this regard, smart and innovative nano-based technologies and approaches encompassing applications of green nanomedicine, bio-inspired methods, multifunctional bioengineered nanomaterials, and biomimetic drug delivery systems/carriers can help resolve the critical issues regarding detection, prevention, and treatment of viral infections. This perspective review expounds recent nanoscience advancements for the detection and treatment of viral infections with focus on coronaviruses and encompasses nano-based formulations and delivery platforms, nanovaccines, and promising methods for clinical diagnosis, especially regarding SARS-CoV-2.

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Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies

Cited by 22 publications

References 32 publications

Exploring the flap dynamics of the South African HIV subtype C protease in presence of FDA‐approved inhibitors: MD study

Exploring the flap dynamics of the South African HIV subtype C protease in presence of FDA‐approved inhibitors: MD study

A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)

Nanomaterials and Nanotechnology-Associated Innovations against Viral Infections with a Focus on Coronaviruses

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