This chapter details the methodology used to produce diffraction-quality crystals of a number of Reverse Transcriptase (RT) complexes. The chapter includes description of the purification of the enzyme and of a noninhibitory Fab used in some of the crystallization experiments, because the reproducible preparation of high-quality crystals of HIV-1 RT is critically dependent on the protocols used to purify each of these proteins. The protocol used for the purification of Fab 28 yields one of the isoelectric variants. Crystallization of the RT Fab complex is carried out in hanging-drop vapor diffusion experiments at 4° over reservoirs containing 0.5 ml of crystallization solution. The parent HIV-1 reverse transcriptase used in the preparation of all the crystal forms described in this chapter is a mutant RT that has serine substituted for cysteine at amino acid position 280. It is fully active in polymerization and Ribonuclease H (RNase H) activities and is resistant to oxidative inactivation of RNase H. Crystals of RT that contain one or two amino acid substitutions have been prepared in complexes with Fab28 and dsDNA.
All Science Journal Classification (ASJC) codes
- Molecular Biology