Expression and purification of HIV-I p15NC protein in Escherichia coli.
An efficient method for the expression and purification of nucleocapsid precursor protein (p15NC) from HIV-I (BH 10 isolate) was developed and used to obtain large quantities of this viral protein for structural studies, protein biochemistry, and high-throughput screening efforts. We have engineered an existing p15NC clone into a new vector developed 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 Escherichia coli and to purify several milligrams of p15NC to homogeneity. In the current purification scheme, lysis of cell paste followed by a simple three-step FPLC procedure yields about 0.4-0.5 mg of purified p15NC per gram of E. coli cell paste expressing the protein with an overall yield of 45%. The purified p15NC retained its ability to bind full-length HIV-I p15NC mRNA in solution- or solid-phase-based assays. A specific stem-loop forming RNA fragment (24-mer) and its antisense DNA oligomer (21-mer) derived from the full-length p15NC mRNA were also able to bind to p15NC. In addition, antisense DNA oligos with bulky 5-iodouracil and 5-iodocytidine substituents were able to bind to p15NC with little or no perturbations as assessed by their ability to compete with 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.
Ozturk
D H
DH
Virology Department, The DuPont Merck Pharmaceutical Company, Experimental Station, Building E336/1B, Wilmington, Delaware, 19880-0336, USA. derya.h.ozturk@dupontpharma.com
Erickson-Viitanen
S
S
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015683:Gene Products, gag / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005814:Genes, Viral; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D019590:Nucleocapsid Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D054301:gag Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015683:Gene Products, gag; D019590:Nucleocapsid Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060295:p15 gag protein, Human immunodeficiency virus 1