Abstract
We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4622-4626). From this procedure we can obtain 100 mg quantities of protein. The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight. Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation. No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence. Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively. The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl. Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.
Highlights
We have purified the /3 subunit of the DNA polym- lead to an understandingof those special features that distinerase I11 holoenzyme to homogeneity producing strain (Blanar, M., Sandler, from an overs., Armengod, M., Ream, L., and Clark, A. (1984) Proc.Natl
The /3 isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity andmolecular weight.Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN
No processing that removes amino acid residues from /3 occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence
Summary
XI1 was Bacteria lS trainsand Grorth-E. coli JC 1357w6hich rapidl yc hangedt o1 0 m M sodium acetate-0.25 DM EDTAby filtration through a Sephadex 0-25 column Cells were grown to late log phase m l SP-Sephadex (10% of the final column volume) that had been ( A s 0 0 = 4 ) at 30'C in 190 1 of media as described (McRenry, 1982). 7.0 and 7.4 by the addition of concentrated KOfl. Overproduction washed with 2 column volume. Of Buffer C and the activity was o f 8 waa induced by inactivation of the temperature-sensitive A eluted with a pfl gradient Fraction IV ( 2 3 4 0 m1; pfl 6.25)
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