Expression, purification, and RNA-binding properties of HIV-1 p15gag nucleocapsid protein.
We have cloned and expressed HIV-1 gag p15 nucleocapsid protein (NCp15) in the form of a 41-kDa fusion polypeptide with glutathione-S-transferase (GST-NCp). The recombinant protein was rapidly degraded in bacterial lysates unless Zn2+ and Cd2+ were present in the extraction buffer. Inclusion of these metals stabilized the protein, allowing facile purification of GST-NCp by affinity chromatography. The native NCp15 was readily prepared from GST-NCp by proteolytic cleavage with thrombin. Both GST-NCp and the processed NCp15 were able to bind RNA containing sequences from the 5'-end of the HIV-1 genome. This binding was unaffected by the absence or the presence of Zn2+; however, the binding of RNA was absolutely dependent on the presence of K+. The GST-NCp fusion protein was nonselective in the binding of RNA, with all transcripts, including antisense and non-HIV RNA, binding with equal efficiency. In contrast, NCp15 was highly selective in binding of RNA. Sequences within nucleotides 1244-1412 of the HIV-1 proviral genome were found necessary for maximal binding of RNA to NCp15.
Li
X
X
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
Gu
Z
Z
Geleziunas
R
R
Kleiman
L
L
Wainberg
M A
MA
Parniak
M A
MA
D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D012367:RNA, Viral / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus
D002412:Cations; D004279:DNA, Viral; D015683:Gene Products, gag; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060295:p15 gag protein, Human immunodeficiency virus 1; D005982:Glutathione Transferase