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

Abstract

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...

The Human Immunodeficiency Virus Type 1 Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation I132M Confers Hypersensitivity to Nucleoside Analogs

“…WT and mutant recombinant HIV-1 RTs were expressed and purified to homogeneity as described previously (12,13). For subunit-selective mutagenesis, the p66 and p51 RT genes were cloned into the pET-DUET vector (Novagen-EMD Biosciences Inc., San Diego, CA) and enzymes were purified using a double-tag strategy as described previously (14,15). The RT concentration was determined spectrophotometrically at 280 nm by using an extinction coefficient (ε 280 ) of 260,450 M Ϫ1 cm Ϫ1 .…”

Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase

“…The C-terminal tag was appended as described [clone 3 (27)]. Glutathione S-transferasetagged p51 (GST-p51) was prepared by subcloning the BamHI-SalI fragment from p51HXGADNOT into pGEX5X-3 (Amersham Pharmacia).…”

A New Method for Protein Coexpression in Escherichia coli Using Two Incompatible Plasmids

“…The other strategy relies on the two-plasmid coexpression system in which foreign genes are cloned in two different vectors (20)(21)(22)(23)(24)(25). Due to its wide application and high copy number, a ColE1-derived plasmid is the most attractive candidate.…”