Cloning, expression, purification, and crystallisation of HIV-2 reverse transcriptase.
A purification procedure is described for the isolation of recombinant HIV-2 reverse transcriptase expressed in Escherichia coli. The p68 subunit is expressed, in the absence of induction, and use of a heparin-Sepharose column produces substantially pure protein. Concentration of the homodimeric p68 reverse transcriptase pool, followed by incubation at room temperature for several days, results in full conversion by E. coli proteases to the heterodimer (p68/p55). This extended incubation simplifies the purification process and improves the yield of heterodimeric reverse transcriptase, which shows a truncation of the smaller subunit to 427 residues. The protein is then purified further by hydroxyapatite and gel-filtration chromatography to homogeneity. The HIV-2 RT is active and has been used to produce crystals that diffract to beyond 3.0 A.
Bird
Louise E
LE
Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, Henry Wellcome Building of Genomic Medicine, University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7BN, UK.
Chamberlain
Philip P
PP
Stewart-Jones
Guillaume B E
GB
Ren
Jingshan
J
Stuart
David I
DI
Stammers
David K
DK
D003001:Cloning, Molecular; D003460:Crystallization; D015870:Gene Expression; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D013058:Mass Spectrometry; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase