Use of PEG to acquire highly soluble DNA-packaging enzyme gp16 of bacterial virus phi29 for stoichiometry quantification.
All linear dsDNA viruses package their genome into a preformed procapsid via a ATP-driving motor involving two nonstructural enzymes or ATPase. This essential viral replication step has been investigated in the quest for new antiviral drugs. These DNA-packaging motors could be potential parts in nanotechnology. But both the low solubility and self-aggregation of all nonstructural enzymes have seriously hampered studies on these motors. Bacterial virus phi29 DNA-packaging motor has been well characterized. But the role of the nonstructural ATPase gp16 has not been well defined due to its hydrophobicity, low solubility, and self-aggregation. Here we report a novel approach to obtain affinity-purified, soluble, and highly active native gp16 with the aid of polyethylene glycol (PEG) or acetone. With several thousand-fold increase in specific activity in comparison to the traditional method, this unique approach has made the quantification of gp16 feasible. The basic functional unit of gp16 in solution was found to be a monomer, as determined by sedimentation and size exclusion chromatography. This result leads to a subsequent finding that the stoichiometry of gp16 for phi29 DNA-packaging was about 11+/-2. These findings will facilitate the study on this novel motor that involves three pRNA dimers and a 12-subunit connector.
Huang
Lisa P
LP
Department of Pathobiology, Purdue Cancer Research Center, Purdue University, Hansen Life Science Research Building B-36, West Lafayette, IN 47907, USA.
Guo
Peixuan
P
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D017103:Bacillus Phages / Q000201:enzymology*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D011092:Polyethylene Glycols / Q000494:pharmacology*; D013696:Temperature; D014764:Viral Proteins / Q000302:isolation & purification*; D019065:Virus Assembly
D004279:DNA, Viral; D014764:Viral Proteins; D011092:Polyethylene Glycols; D000251:Adenosine Triphosphatases