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

BACKGROUND: Human 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. RESULTS: A 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. CONCLUSIONS: By 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.

Two-Step Preparation of Highly Pure, Soluble HIV Protease from Inclusion Bodies Recombinantly Expressed in Escherichia coli.

Heterologous expression of exogenous proteases in Escherichia coli often results in the formation of insoluble inclusion bodies. When sequestered into inclusion bodies, the functionality of the proteases is minimized. To be characterized structurally and functionally, however, proteases must be obtained in their native conformation. HIV protease is readily expressed as inclusion bodies, but must be recovered from the inclusion bodies. This protocol describes an efficient method for recovering HIV protease from inclusion bodies, as well as refolding and purifying the protein. HIV protease-containing inclusion bodies are treated with 8 M urea and purified via cation-exchange chromatography. Subsequent refolding by buffer exchange via dialysis and further purification by anion-exchange chromatography produces highly pure HIV protease that is functionally active. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Recovery, refolding, and purification of HIV protease from inclusion bodies Support Protocol 1: Expression and extraction of inclusion bodies containing HIV protease expressed in Escherichia coli Support Protocol 2: Determination of the active site concentration of HIV protease via isothermal titration calorimetry.

In vitro translation to study HIV protease activity.

HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for virus replication. Here, we describe an in vitro transcription/translation-based method of phenotyping HIV-1 PR. In this system, both substrate and PR for the assay can be prepared by in vitro transcription/translation. Protease activity is estimated by the cleavage of a substrate, as measured by enzyme-linked immunosorbent assay (ELISA). This assay is safe, rapid, and requires no special facility to be carried out. Our rapid phenotyping method of HIV-1 PR may help evaluate drug resistance, useful when choosing an appropriate therapeutic regiment, and could potentially facilitate the discovery of new drugs effective against HIV-1 PR.

Establishment of a new cell line inducibly expressing HIV-1 protease for performing safe and highly sensitive screening of HIV protease inhibitors.

The human immunodeficiency virus type 1 (HIV-1) protease (PR) plays an essential role in processing viral polyproteins into mature proteins. As a result, it is a major target for the development of drugs against AIDS. However, due to the rapid emergence of drug-resistant HIV, the development of novel HIV PR inhibitors is urgently needed. We recently established a new cell line E-PR293 which can be used as a safe, convenient and highly efficient assay system to screen HIV-1 PR inhibitors. In the cells, the HIV-1 PR is expressed in a chimeric protein with the green fluorescence protein (GFP). This assay measures the PR activity as a function of either the fluorescence of GFP or the cytotoxic activity of HIV-1 PR which is expressed in the cell. E-PR293 cells were maintained in the presence of doxycycline, which suppresses the expression of HIV-1 PR. The removal of doxycycline induces the expression of HIV-1 PR, which is used to screen HIV-1 PR inhibitors. In E-PR293 cells, the 50% inhibitory concentration of the cytotoxic effects by nelfinavir and saquinavir were as low as nanomolar levels, almost equal to those found in the HIV-infection assay.

In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling.

BACKGROUND: Given the expanding antiretroviral therapy, inexpensive and fast HIV drug resistance assays are urgently needed. In this view, we have developed a novel phenotypic resistance test for HIV-1 protease inhibitors (PIs) based on recombinant expression of patient-derived HIV PR in Escherichia coli and subsequent enzymatic testing in a fluorescent readout. OBJECTIVES: To facilitate and expedite the test procedure, we have introduced coupled in vitro transcription/translation using a commercially available technology called RTS for producing enzymatically active HIV-1 protease (PR). STUDY DESIGN: We expressed one wild type PR and one highly resistant mutant starting from molecular clones as well as three patient-derived PRs. The amplified PR gene was either ligated into an expression vector or directly used as a template for the in vitro transcription/translation reaction. Enzymatic susceptibility data derived from in vitro expressed PRs were correlated to the respective results from E. coli expression and genotypic evaluation. RESULTS: All tested enzymes were obtained in sufficient quantities for complete resistance profiling to five PIs. The PRs required no purification prior to the enzymatic assay. Inhibition constants and enzymatic resistance factors compared well to corresponding data from PRs expressed in parallel in E. coli. Enzymatic resistance was in good agreement with the respective PR genotype. CONCLUSION: The presented in vitro transcription/translation system represents a novel approach for HIV PR expression starting from molecular clones or patient samples. Coupled with the enzyme-kinetic PR assay recently developed in our group it allows to sensitively quantify resistance to PIs. The test system is significantly less laborious and faster than currently available phenotypic drug resistance assays.

Non-infectious in-cell HIV-1 protease assay utilizing translocalization of a fluorescent reporter protein and apoptosis induction.

This study describes a non-infectious in-cell imaging assay for HIV-1 protease inhibitor screening. It is based on re-distribution of a fluorescence reporter protein upon protease cleavage and the fact that HIV-infected cells undergo apoptosis. The in-cell assay utilizes fluorescent reporter proteins consisting of an intracellular translocation signal sequence, a caspase-3-specific cleavage sequence, and a fluorescent tagging protein. The reporter proteins are designed to change their intracellular localization upon cleavage, either from the cytosol to a subcellular organelle (type I) or from a subcellular organelle to the cytosol (type II). Inhibition of protease activity can be monitored at the single cell level. Interestingly, the expression of HIV-1 protease induced endogenous caspase-3 activation; thus, the fluorescence reporter protein containing the caspase-3 cleavage sequence translocalized upon cleavage. This is the first time that HIV-1 protease expression, not whole virus infection of the cell, was observed to trigger the apoptotic pathway, including caspse-3 activation. A validation of this assay was performed with a known HIV-1 protease inhibitor, Ac-Leu-Val-phenylalanine. The clear cellular change in fluorescence pattern makes this system an ideal tool for various types of life science and drug discovery research, including high throughput and high content screening applications.

High-level production of active HIV-1 protease in Escherichia coli.

High levels of active HIV-1 protease (PR) were produced in Escherichia coli, amounting to 8-10% of total cell protein. High production levels were achieved by altering the following parameters: (1) codon preference of the coding region, (2) A+T-richness at the 5' end of the coding region, and (3) promoter. To circumvent the toxicity of HIV-1 PR in E. coli, the gene was expressed as a fusion protein with two different proteolytic autocleavage sequences. In both the cases, the fusion protein could be cleaved in vivo to give an active molecule with the native sequence at the N terminus.

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold.

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.

Analysis of HIV type 1 reverse transcriptase expression in a human cell line.

The functional analysis of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) subunits on transient and constitutive expression, in the absence or presence of the HIV-1 protease (PR) expression, in a human cell line is described. HIV-1 RT is a heterodimer composed of a 51-kDa subunit (p51) and a 66-kDa subunit (p66). Cloning and expression of the RT region of the HIV-1 pol gene in the HT-1080 human fibrosarcoma cell line yielded p66 without any detectable p51 and a low level of RT activity could be measured. Transient expression of PR and RT in cis generated p51 and p66, but when RT and PR were expressed in trans only p66 was produced. Attempts to establish a stable cell line expressing the PR-RT region of the pol gene were hampered by an apparent intolerance of HT-1080 cells to the HIV-1 PR expression. Therefore, to generate p51 independent of PR expression, the 51-kDa subunit was cloned separately. p51 lacked detectable RT activity. Coexpression of p51 and p66 resulted in a dramatic increase in RT activity. Stable HT-1080 cells producing both p51 and p66 exhibited on average a 15-fold increase in RT activity compared to the parental cell line. Immunofluorescence revealed a diffuse cytoplasmic localization of p51 and p66. To date, this is the first example of a human cell line that is constitutively expressing HIV-1 RT in the absence of HIV-1 infection.

Characterization of HIV replication complexes early after cell-to-cell infection.

In this study, we have characterized the HIV DNA-containing replication complexes present in cells early after cell-to-cell infection, using sucrose gradient sedimentation and immunoprecipitation. Six hours after cell-to-cell infection, a cytoplasmic HIV replication complex sedimented as a large structure (320S). This replication complex was precipitated by antisera to three virus-coded enzymes (reverse transcriptase, integrase, protease), to the matrix protein (p17), and to cellular histones but not to the major capsid protein (p24). This replication complex was not associated with cell membranes and could not be dissociated into smaller discrete subunits, using detergents. Nuclear extracts from the same cell-to-cell infection contained a smaller (80S) complex that lacked reverse transcriptase and matrix protein (p17). Cytoplasmic replication complexes from a cell-free virus infection sedimented as 160S structures under identical conditions, as previously reported. Our results indicate that, following cell-to-cell transmission of HIV, all the HIV pol gene products, the matrix protein p17, and cellular histones are present in cytoplasmic replication complexes that are taking part in or have completed reverse transcription. Transportation of the cytoplasmic replication complex to the nucleus is associated with structural changes, including a reduction in size and altered protein composition.

Intrinsic activity of human immunodeficiency virus type 1 protease heterologous fusion proteins in mammalian cells.

We have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein.

Large scale expression and purification of recombinant HIV-1 proteinase from Escherichia coli.

The availability of target proteins in sufficient quantity is a limiting factor in crystallographic studies and therefore in rational drug design. Even after optimisation, expression of recombinant proteins may be low and the only way to produce enough protein is by large scale cell growth/purification. HIV-1 proteinase in Escherichia coli, which due to its toxicity is expressed as a soluble protein only at around 0.1% of total protein, is a paradigm for this. In this paper a detailed process for large scale expression and purification of HIV-1 proteinase which delivers material of suitable quantity (30 mg from 500 g of wet weight of cells) and quality for crystallographic studies is described.

Molecular characterization of HIV-2 (ROD) protease following PCR cloning from virus infected H9 cells.

A 450 nucleotide sequence corresponding to the nucleotides 1931-2380 of the viral genome (8) was amplified by polymerase chain reaction (PCR) using template DNA prepared from HIV-2 (ROD) infected H9 cells. The sequence codes for HIV-2 protease and its N-terminal flanking peptide. An identical DNA sequence was obtained from three independent PCR amplifications, which differs from the published sequence of HIV-2 (ROD) in 7 nucleotides scattered throughout the region of the cloned DNA. The cloned DNA was expressed in E. coli cells and resulted in the synthesis of a correctly processed HIV-2 protease, which is enzymatically active. Therefore, none of the seven nucleotide changes, which resulted in two amino acid substitutions, affect the autoproteolytic or trans-cleaving activities of the HIV-2 protease.

[Establishment of a microbial assay for screening anti-human immunodeficiency virus type-1 protease inhibitors].

OBJECTIVE: This study was to establish a microbial assay of human immunodeficiency virus type-1 protease (HIV-1 PR) activity for screening anti-HIV PR inhibitors. METHODS: A 24 bp synthetic oligonucleotide fragment that encodes the HIV-1 PR recognition sequence was inserted into the tetr gene of pBR322 (mtetr). Escherichia coli containing HIV-1 PR expression vector-pPOLO was transformed with pACYC184M containing modified mtetr gene. The transformant could express both HIV-1 PR and the modified Tet protein. RESULTS: The growth of engineered E. coli was prevented in the presence of tetracycline because the resistance Tet protein was degraded by HIV-1 PR. However inhibition of the HIV-1 PR restored tetracycline resistance. 31 chemical synthetic compounds were tested by the microbial assay. CONCLUSIONS: A microbial assay method of HIV-1 PR activity was established through a engineered E. coli. 5 mumol/L saqunavir-a special HIV-1 PR inhibitor showed inhibitory effect on the engineered E. coli. That means this model could be used as a initial screening model for anti-HIV PR agents.

[The properties of HIV protease synthesized in E. coli cells]. / Izuchenie svoistv proteazy VICh, sintezirovannoi v kletkakh bakterii E. coli.

A hybrid plasmid pPR6 was constructed containing BgII-EcoRI fragment of the pol region of HIV (strain IIIB) genome which determined the synthesis of virus-specific protease. Extracts of E. coli DN5/pPR6 bacteria provided for specific hydrolysis of hybrid protein p165 (the N-terminus of which is presented by complete beta-galactosidase and the C-terminus by duplicated area of virus-specific precursor p55 containing a site for virus-specific protease located at the border of proteins p17 and p24) with formation of products having molecular weights of 19, 42, 28, 23, and 19 kD. Polypeptides 119K, 23K, and 19K are products of complete hydrolysis, and 42K a result of partial cleavage. The kinetics of hydrolysis in relation to pH values of the reaction mixture was analysed. It is suggested that the reported system of HIV protease activity determination be used for screening of potential inhibitors of this enzyme.

Yeast cells as a tool for analysis of HIV-1 protease susceptibility to protease inhibitors, a comparative study.

HIV develops drug resistance at a high rate under drug selection pressure. Resistance tests are recommended to help physicians optimize antiretroviral drug therapies. For this purpose, genotypic and phenotypic tests have been developed. In order to propose a new phenotypic test that will be less laborious, expensive, and time consuming than the standard ones, a new procedure to measure HIV-1 protease susceptibility to protease inhibitor (PIs) in Saccharomyces cerevisiae yeast cells was developed. This procedure is based on HIV-1 protease expression in yeast. While the viral protein induces yeast cell death, its inhibition by PIs in the culture medium allows the cell to grow in a dose-dependent manner. In a comparative study of standard genotypic analysis vs. yeast cell-based phenotypic tests, performed on HIV-1 protease coding DNA in 17 different plasma samples from infected individuals, a clear match was found between the results obtained using the two technologies. This suggests that the yeast-based procedure is at least as accurate as standard genotypic test in defining susceptibility to protease inhibitors. This encouraging result should be the basis for large-scale validation of the new phenotypic resistance test.

Increased expression and immunogenicity of HIV-1 protease following inactivation of the enzymatic activity.

HIV-1 protease is an important target for anti-HIV therapy but has not received much attention as a vaccine antigen. To investigate the immunogenic properties of HIV-1 protease, we designed DNA plasmids encoding variants of the protease gene. Mutations resulting in enzymatic inactivation (D25N) and resistance to standard antiretroviral drugs (V82F/I84V) were introduced in order to examine the impact of the enzymatic activity on immunogenicity and the possibility to induce immune responses against drug resistant protease, respectively. The enzymatic inactivation of protease resulted in significantly increased in vitro expression as well as in vivo immunogenicity. The inactivated protease was highly immunogenic in both BALB/c and HLA-A0201 transgenic C57Bl/6 mice, and the immunogenicity was retained when the gene was delivered as a part of a multigene HIV-1 DNA vaccine. The drug resistance mutations hampered both the cellular and humoral immune responses, as the mutations also affect both CD4 and CD8 T cell epitopes. Taken together, our data demonstrates the possibility to drastically increase the immunogenicity of HIV-1 protease.