Production of active mouse DNA polymerase delta in bacteria.
The entire cDNA encoding the large subunit of mouse DNA polymerase delta (mPol delta; EC 2.7.7.7) has been cloned and expressed in various bacterial expression systems. A soluble protein could only be obtained when mPol delta was produced as a glutathione S-transferase (GST) fusion protein and the incubation temperature of the expression strain was reduced to 30 degrees C. After purification over a glutathione-Sepharose column, the fractions containing the recombinant (re-) fusion protein showed both DNA Pol and 3'-->5' Exo activities. In situ activity gel analysis indicated that the Pol activity resides in the re-protein. This activity, however, was not stimulated by proliferating cell nuclear antigen (PCNA). Our data are discussed in the view of the findings of Goulian et al. [J. Biol. Chem., 265 (1990) 16402-16411] that the second mPol delta subunit, the 48-kDa protein, might play an important role in DNA Pol delta-PCNA interaction.
Hindges
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Institut für Veterinärbiochemie, Universität Zürich-Irchel, Switzerland.
Hübscher
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U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004258:DNA Polymerase III; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase