Refolding active human DNA polymerase nu from inclusion bodies.
Human DNA polymerase nu (Pol nu) is a conserved family A DNA polymerase of uncertain biological function. Physical and biochemical characterization aimed at understanding Pol nu function is hindered by the fact that, when over-expressed in Escherichia coli, Pol nu is largely insoluble, and the small amount of soluble protein is difficult to purify. Here we describe the use of high hydrostatic pressure to refold Pol nu from inclusion bodies, in soluble and active form. The refolded Pol nu has properties comparable to those of the small amount of Pol nu that was purified from the soluble fraction. The approach described here may be applicable to other DNA polymerases that are expressed as insoluble inclusion bodies in E. coli.
Arana
Mercedes E
ME
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Powell
Gary K
GK
Edwards
Lori L
LL
Kunkel
Thomas A
TA
Petrovich
Robert M
RM
D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D017510:Protein Folding; D020673:Protein Renaturation; D012995:Solubility
D004259:DNA-Directed DNA Polymerase; C546205:POLN protein, human