Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up.
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
Maier
G
G
Max-Planck-Institut für Biochemie, Martinried, Germany.
Dietrich
U
U
Panhans
B
B
Schröder
B
B
Rübsamen-Waigmann
H
H
Cellai
L
L
Hermann
T
T
Heumann
H
H
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D006639:Histidine; D054303:HIV Reverse Transcriptase