High-level expression and purification of untagged and histidine-tagged HIV-1 reverse transcriptase.
We have devised simplified protocols to purify large quantities of histidine-tagged (His-tagged) and untagged heterodimeric forms of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). Here, we report the optimization of overexpression and purification of heterodimeric RT expressed in Escherichia coli. The coding sequences of p66 and p51 subunits of RT were amplified using PCR from HXB2 HIV-1 and cloned into a bacterial expression system. The resulting expression plasmids for the RT subunits, pET-RT66 and pET-RT51, were under a strong T7/lac promoter that is induced by isopropyl-beta-d-thiogalactopyranoside. Purification of heterodimeric forms of RT was facilitated by high-level expression of these subunits that represented approximately 30-40% of total cell protein. For purification of the His-tagged heterodimeric RT, cell pellet from cells expressing the untagged p66 subunit was mixed in excess with a cell pellet expressing tagged p51. For untagged heterodimeric RT, the pellet from cells expressing p51 was mixed in excess with pellet expressing p66. Subunit dimerization occurred during cell lysis. During the subsequent chromatography steps, stable p66/p51 heterodimer was purified to homogeneity. The heterodimeric nature of the final preparations of RT was confirmed by analytical gel filtration, mass spectrometry, and denaturing gel electrophoresis. Further, the sensitivity of these enzyme preparations to AZTTP indicated that the histidine tag had no effect on nucleoside inhibitor binding, nucleotide binding or insertion, or DNA binding. The application of these expression/purification methodologies represents a useful method to purify large quantities of heterodimeric RT for structural investigations and provides an efficient protocol to produce subunit-specific amino acid alterations necessary for unambiguous structure/function investigations.
Hou
Esther W
EW
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Prasad
Rajendra
R
Beard
William A
WA
Wilson
Samuel H
SH
D019380:Anti-HIV Agents; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D054306:Dideoxynucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013942:Thymine Nucleotides / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000494:pharmacology
D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D015215:Zidovudine; D006639:Histidine; C052873:zidovudine triphosphate; D054303:HIV Reverse Transcriptase