PMC

Diffraction from a crystal of the Pfu Pol–PfuPCNAm complex. The arrow indicates a diffraction spot at 3.1 Å resolution.

(a) Crystals obtained from the precipitant solution of ammonium sulfate and polyethylene glycol monomethylether 5000. (b) The addition of 1%(v/v) 1,8-diaminooctane to the above solution dramatically improved the crystal size and inhibited the clustering.

The elution profiles of PfuPolBI with PfuPCNAm gel-filtration chromatography measured at 260 and 280 nm are indicated by the elution volume; they are compared with those of purified PfuPolBI. The elution positions of the marker proteins are shown by arrowheads, with each molecular weight (in kDa) under the chromatographic profile. Each peak fraction was subjected to SDS–PAGE analysis as shown in Fig. 3.

5 µl aliquots of the peaks from the gel-filtration analysis and a dissolved sample of the obtained crystals were analyzed by SDS–PAGE. The gel was stained with Coomassie blue dye. Lane 1, molecular-weight markers (sizes are shown in kDa on the left); lane 2, PfuPolBI; lane 3, PfuPolBI + PfuPCNAm, gel filtration; lane 4, PfuPolBI + PfuPCNAm, crystal. The crystals were washed in the precipitant solution and then dissolved in buffer A.