Table 1.

Purification of RNR from T. acidophila

Step	Activity, nmol/h	Volume, ml	Total protein, mg	Specific activity, nmol/h·mg	Yield, %	Purification factor
Crude extract	2050	22	246	8.3	100	1
PEG precipitation	1590	10	115	13.8	78	1.7
Q-Sepharose	2310	40	41	56	113	6.7
(NH4)2SO4 precipitate	2160	5	11.3	191	105	23
BioGel HTP	2060	0.5	4.48	460	100	55
HPLC sizing	255	2.1	0.33	773	12.4	93
HPLC-DEAE	109	2.75	0.12	908	5.3	110

From a 10-liter culture of T. acidophila in Tris·HCl buffer (100 mM, pH 8.0, 3°C). Sonicated extracts from 2 g of cells were fractionated (4-8%, wt/vol) with polyethylene glycol (PEG) 8000. The enriched protein was loaded onto a 150-ml Q-Sepharose column and eluted with a linear gradient of NaCl (0-1 M). After fractionation [30% saturation (NH₄)₂SO₄], protein was loaded onto a 25-ml BioGel HTP column and eluted with a linear gradient of potassium phosphate (0-0.25 M, in 100 mM Tris·HCl, pH 8.0). RNR was precipitated [30-55% saturated (NH₄)₂SO₄], and the concentrated protein was purified by HPLC size exclusion chromatography (LKB TSK-G3000, 8 × 300 mm, room temperature).