Single-step purification of recombinant wild-type and mutant HIV-1 reverse transcriptase.
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.
Fletcher
R S
RS
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis- Jewish General Hospital, Montreal, Canada.
Holleschak
G
G
Nagy
E
E
Arion
D
D
Borkow
G
G
Gu
Z
Z
Wainberg
M A
MA
Parniak
M A
MA
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
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