反应详情

The thus-obtained cell was suspended in 600 ml of a 0.1M phosphate buffer solution, and broken by the use of a Dyno-mill. After the removal of insoluble materials by centrifugal separation, a crude extract containing adenylate kinase was obtained. Then, 200 ml of a 10% streptomycin sulfate solution was added to 400 ml of the crude extract. Precipitates formed were removed by centrifugal separation to thereby obtain a streptomycin supernatant liquid. This supernatant liquid was subjected to ammonium sulfate fractionation, and a fraction from 30% saturation (4° C.) to 60% saturation (4° C.) was obtained. This fraction was dissolved in a 50 mM Tris-hydrochloric acid buffer solution (pH 8.0) and passed through a DEAE-Sephadex column which had been equilibrated with the same buffer solution as above, and thereafter it was eluted with a solution prepared by adding sodium chloride to the above buffer solution. Near a concentration of sodium chloride of 0.2M, the desired adenylate kinase was eluted. The thus-eluted fraction was subjected to hydroxyapatite column chromatography under the same conditions as in Example 1, passed through a Sephadex G-75 column, and eluted with a 30 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 0.1M sodium chloride to thereby obtain an adenylate kinase sample which provided a single band by acrylamide disc electrophoresis as in the case of Example 1. Furthermore, as in the case of Example 1, the adenylate kinase sample provided a single peak at a molecular weight of about 22,000 as determined by Sephadex G-100 chromatography.

1. customAfter the removal of insoluble materials
2. customby centrifugal separation
3. extractiona crude extract
4. additioncontaining adenylate kinase
5. customwas obtained
6. extractionextract
7. customPrecipitates
8. customformed
9. customwere removed by centrifugal separation