A two plasmid co-expression system in Escherichia coli for the production of virion-like reverse transcriptase of the human immunodeficiency virus type 1

Jonckheere, Heidi; Vreese, K De; Debyser, Zeger; Vandekerckhove, Joël; Balzarini, Jan; Desmyter, Jan; Clercq, Erik De; Anné, Jozef

doi:10.1016/0166-0934(96)02076-9

Cited by 21 publications

(19 citation statements)

References 28 publications

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“…Recombinant HIV-1 RT enzymes were expressed from a twoplasmid coexpression system as previously described [15]. The p66 subunit of RT was expressed from pACYC66His and the p51 subunit from pKRT51.…”

Section: Construction Of Mutant Recombinant Hiv-1 Reverse Transcriptasesmentioning

confidence: 99%

“…In the case, where d4TTP (or PFA) were evaluated for their inhibitory activity, 0.15 mM poly(rA)AEoligo(dT) [12][13][14][15][16][17][18] was used as the template/primer, and 1.6 lM [ 3 H]dTTP as the radiolabeled substrate. The reaction mixtures were incubated at 37°C for 30 min, at which time 200 ll of yeast RNA (2 mg/ml) and 1 ml of trichloroacetic acid (TCA) (5% in 20 mM Na 4 P 2 O 7 ) were added.…”

Section: Reverse Transcriptase Assaymentioning

confidence: 99%

“…For determination of the 50% inhibitory concentration (IC 50 ) of the test compounds against HIV-1 RT, the RNA-dependent DNA polymerase assay was performed as follows: the reaction mixture (50 ll) contained 50 mM Tris-HCl (pH 7.8), 0.06% Triton X-100, 5 mM DTT, 0.3 mM glutathione, 150 mM KCl, 5 mM MgCl 2 , 1.25 mg/ml BSA, 0.5 mM EDTA, 0.1 mM template/primer poly(rC)AEoligo(dG) [12][13][14][15][16][17][18] (Amersham Biosciences), a fixed concentration of the labeled substrate [8-3 H]dGTP (1.6 lM, 1 lCi; specific activity, 12.6 Ci/mmol; Amersham Biosciences), 5 ll of inhibitor solution [containing various concentrations (10-fold dilutions) of the compounds], and 5 ll of the RT preparations. In the case, where d4TTP (or PFA) were evaluated for their inhibitory activity, 0.15 mM poly(rA)AEoligo(dT) [12][13][14][15][16][17][18] was used as the template/primer, and 1.6 lM [ 3 H]dTTP as the radiolabeled substrate.…”

Section: Reverse Transcriptase Assaymentioning

confidence: 99%

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The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti‐HIV‐1 drugs

et al. 2005

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The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti-HIV-1 drugs Abstract Amino acids N137 and P140 in the p51 subunit of HIV-1 reverse transcriptase (RT) are part of the b7-b8-loop that contributes to the formation of the base of the non-nucleoside RT inhibitor (NNRTI)-binding pocket and makes up a substantial part of the dimerization interface. Amino acid P95 in p66 also markedly contributes to the dimerization binding energy. Nine RT mutants at amino acid 137 were constructed bearing the mutations Y, K, T, D, A, Q, S, H or E. The prolines at amino acid positions 95 and 140 were replaced by alanine in separate enzymes. We found that all mutant RT enzymes showed a dramatically decreased RNA-dependent DNA polymerase activity. None of the mutant RT enzymes showed marked resistance against any of the clinically used NNRTIs but they surprisingly lost significant sensitivity for NRTIs such as ddGTP. The denaturation analyses of the mutant RTs by urea are suggestive for a relevant role of N137 in the stability of the RT heterodimer and support the view that the b7-b8 loop in p51 is a hot spot for RT dimerization and instrumental for efficient polymerase catalytic activity. Consequently, N137 and P140 in p51 and P95 in p66 should be attractive targets in the design of new structural classes of RT inhibitors aimed at compromising the optimal interaction of the b7-b8 loop in p51 at the p66/p51 dimerization interface.

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Section: Construction Of Mutant Recombinant Hiv-1 Reverse Transcriptasesmentioning

confidence: 99%

Section: Reverse Transcriptase Assaymentioning

confidence: 99%

Section: Reverse Transcriptase Assaymentioning

confidence: 99%

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The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti‐HIV‐1 drugs

et al. 2005

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“…Moreover, the C-terminus of p51 can be degraded by bacterial proteases with an average loss of seven residues [8]. Deletion of 13 residues at the C-terminus of p51 was reported to impair RT functions dramatically [9].Among the existing reconstitution strategies, the most straightforward ones are selection of N-terminally His-tagged p51 against the p51 derived from p66 degradation [10,11] and the use of coexpression systems in which in vivo heterodimer formation protects p66 from degradation [12,13].We constructed a new coexpression system, where the two subunits are expressed in the same cells from two compatible commercially available plasmids. The system described here incorporates the advantages of the best described techniques, while introducing the GST-fusion approach for selection of desired products and a procedure that allows the removal of excess of p51, including its truncated forms, by hydrophobic chromatography.…”

mentioning

confidence: 99%

“…Among the existing reconstitution strategies, the most straightforward ones are selection of N-terminally His-tagged p51 against the p51 derived from p66 degradation [10,11] and the use of coexpression systems in which in vivo heterodimer formation protects p66 from degradation [12,13].…”

mentioning

confidence: 99%

Mixed reconstitution of mutated subunits of HIV‐1 reverse transcriptase coexpressed in Escherichia coli – two tags tie it up

Maier

Dietrich

Panhans

et al. 1999

European Journal of Biochemistry

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The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding Keywords: HIV-1 reverse transcriptase; heterologous expression; GST-fusion; coexpression; helix-clamp motif.The reverse transcriptase (RT) of HIV-1, the causative agent of AIDS, catalyzes the synthesis of the proviral dsDNA, a crucial step in viral replication [1,2]. Replication starts with the synthesis of minus-strand DNA initiated from the 3 H -end of the host cellular tRNA Lys3 , which is complementary to a sequence of the viral ssRNA. The synthesis of proviral dsDNA therefore requires an RNA-dependent polymerase activity as well as an RNase H activity to degrade the copied genomic RNA template. Initation of plus-strand DNA synthesis from the so-called polypurine tract requires a DNA-dependent polymerase activity. Thus, during replication the RT needs to bind diverse primer/template substrates such as dsRNA, RNA/DNA, DNA/RNA and dsDNA.RT consists of two subunits, where the smaller p51 subunit is generated from C-terminal proteolytic cleavage of the larger p66 subunit by viral protease [3]. Although both subunits harbour the same N-terminus, the crystal structure reveals an asymmetric heterodimer in which p66 comprises the polymerase as well as the RNase H active sites [4,5]. It has been suggested that a helixturn-helix structure, termed the helix clamp, is involved in nucleic acid binding in both subunits [6].The aim of this work is to establish a system for obtaining mixed reconstituted RT which consists of selectively mutated subunits in order to analyse t...

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Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens

Kim

Jeong

et al. 2021

Advanced Biology

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Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof‐of‐concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS‐CoV and HCoV‐OC43. Antibodies raised against these antigens, which contain human N‐glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.

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A two plasmid co-expression system in Escherichia coli for the production of virion-like reverse transcriptase of the human immunodeficiency virus type 1

Cited by 21 publications

References 28 publications

The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti‐HIV‐1 drugs

The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti‐HIV‐1 drugs

Mixed reconstitution of mutated subunits of HIV‐1 reverse transcriptase coexpressed in Escherichia coli – two tags tie it up

Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens

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