title: Single-step purification of recombinant wild-type and mutant HIV-1 reverse transcriptase.
abstract: We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h. Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series. The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient. The purification has been carried out with both medium-pressure and high-pressure chromatographic systems. Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity. A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods. The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase. We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance.
authors.affiliation: Lady Davis Institute for Medical Research, Sir Mortimer B. Davis- Jewish General Hospital, Montreal, Canada.
authors.forename: R S
authors.initials: RS
authors.lastname: Fletcher
authors.forename: G
authors.initials: G
authors.lastname: Holleschak
authors.forename: E
authors.initials: E
authors.lastname: Nagy
authors.forename: D
authors.initials: D
authors.lastname: Arion
authors.forename: G
authors.initials: G
authors.lastname: Borkow
authors.forename: Z
authors.initials: Z
authors.lastname: Gu
authors.forename: M A
authors.initials: MA
authors.lastname: Wainberg
authors.forename: M A
authors.initials: MA
authors.lastname: Parniak
mesh_terms: D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D003848:Deoxyguanine Nucleotides; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D011061:Poly A; C018368:poly(rA).oligo(dT); C029603:deoxyguanosine triphosphate; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate