EXPRESSION AND PURIFICATION OF HIV-I P15NC PROTEIN IN ESCHERICHIA-COLI

An efficient method for the expression and purification of nucleocapsi d precursor protein (p15NC) from HIV-I (BH 10 isolate) was developed a nd used to obtain large quantities of this viral protein for structura l studies, protein biochemistry, and high-throughput screening efforts . We have engineered an existing p15NC clone into a new vector develop ed at the University of Heidelberg, Germany. Using PCR, we introduced new restriction sites and a strong ribosome-binding site in the p15NC gene and expressed authentic p15NC protein. Our protocol enabled us to rapidly obtain soluble and highly stable p15NC expressed in Escherich ia coil and to purify several milligrams of p15NC to homogeneity. In t he current purification scheme, lysis of cell paste followed by a simp le three-step FPLC procedure yields about 0.4-0.5 mg of purified p15NC per gram of E. coil cell paste expressing the protein with an overall yield of 45%. The purified p15NC retained its ability to bind full-le ngth HIV-I p15NC mRNA in solution- or solid-phase-based assays. A spec ific stem-loop forming RNA fragment (24-mer) and its antisense DNA oli gomer (al-mer) derived from the full-length p15NC mRNA were also able to bind to p15NC. In addition, antisense DNA oligos with bulky 8-iodou racil and 5-iodocytidine substituents were able to bind to p15NC with little or no perturbations as assessed by their ability to compete wit h the full-length p15NC mRNA in filter-binding competition assays, In addition, RNA-dependent cleavage of the purified p15NC in vitro by HIV -I protease occurred at rates similar to those reported previously. (C ) 1998 Academic Press.