Prasad, Rajendra (NIEHS)
Hou, Ester (NIEHS)
Human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) gene encodes 560 amino acids. In the virus, however, HIV-1 RT occurs as a dimer of two related polypeptides, p66 and p51 subunits at a molar ratio of 1:1. The p51 subunit is derived from a C-terminal proteolytic cleavage of the p66 subunit. This invention describes a simplified protocol to purify large quantities of histidine-tagged and untagged heterodimeric forms of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) from Escherichia coli. The coding sequences of p66 and p51 subunits of RT were placed in expression plasmids under a strong T7-lac promoter that is induced by isopropyl B-D-thiogalactopyranoside. Purification of heterodimeric forms of RT was facilitated by high-level expression of these subunits that represented approximately 30-40% of total cellular proteins. For purification of the histidine-tagged heterodimer RT, cell pellet from cells expression the untagged p66 subunit was mixed in excess with a cell pellet expression tagged p51. For untagged heterodimer 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.
This invention makes available a new and simplified protocols to purify large quantities of homogeneous heterodimeric forms of HIV-1 reverse transcriptase. The preparations were fully characterized for the activity and inhibitor sensitivity, and can be used for structural and kinetic studies. Furthermore, the protocols of expression and purification can be applied to preparing subunit-specific amino acid alterations necessary for structure-function investigations.