Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase.
The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).
Bhikhabhai
R
R
Department of Molecular Biology, Uppsala University, Sweden.
Joelson
T
T
Unge
T
T
Strandberg
B
B
Carlsson
T
T
Lövgren
S
S
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D014961:X-Ray Diffraction
D009842:Oligopeptides; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase