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Title
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Cloning, expression, purification, and crystallisation of HIV-2 reverse transcriptase.
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Authors
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L.E.Bird,
P.P.Chamberlain,
G.B.Stewart-Jones,
J.Ren,
D.I.Stuart,
D.K.Stammers.
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Ref.
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Protein Expr Purif, 2003,
27,
12-18.
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PubMed id
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Abstract
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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.
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