HIV-1 Rev expressed in recombinant Escherichia coli: purification, polymerization, and conformational properties.
The high-level expression of HIV-1 Rev in Escherichia coli is described. Protein in crude bacterial extracts was dissociated from bound nucleic acid with urea. A simple purification and renaturation protocol, monitored by circular dichroism, is described which results in high yields of pure protein. The purified protein binds with high affinity to the Rev-responsive element mRNA and has nativelike spectroscopic properties. The protein exhibits concentration-dependent self-association as judged by analytical ultracentrifugation and gel filtration measurements. Purified Rev showed reversible heat-induced aggregation over the temperature range 0-30 degrees C. This hydrophobic-driven and nonspecific protein association was inhibited by low concentrations of sulfate ions. Rev solutions at greater than 80 micrograms/mL, incubated at 0-4 degrees C, slowly polymerized to form long hollow fibers of 20-nm diameter. Filament formation occurs at a lower protein concentration and more rapidly in the presence of Rev-responsive mRNA. The nucleic acid containing filaments are about 8 nm in diameter and up to 0.4 micron in length. On the basis of physical properties of the purified protein, we have suggested that in the nucleus of infected cells, Rev binding to the Rev-responsive region of env mRNA may be followed by helical polymerization of the protein which results in coating of the nucleic acid. Coated nucleic acid could be protected from splicing in the nucleus and exported to the cytoplasm.
Wingfield
P T
PT
Protein Expression Laboratory, Office of the Director, National Institutes of Health, Bethesda, Maryland 20892.
Stahl
S J
SJ
Payton
M A
MA
Venkatesan
S
S
Misra
M
M
Steven
A C
AC
D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015694:Gene Products, rev / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015752:Genes, env / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D014461:Ultracentrifugation; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D046911:Macromolecular Substances; D012367:RNA, Viral; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus