Table 1.

Purification of TelN

Fraction	Purification step	Protein, mg	Yield, %	Purity, %
I	Precipitation with (NH4)2SO4	2394	100	7
II	Heparin-Sepharose CL-6B	201	83	70
III	Hydroxylapatite Bio-Gel HT	186	77	70
IV	DEAE-Sephacel	61.9	32	90
V	Phosphocellulose P11	11.4	7	96

The purification of N15 TelN was followed by SDS/PAGE before its activity was discovered. Cultures (4 × 1.2 liters) of SCS1(pJD101) were grown at 37°C with shaking. At an A₆₀₀ of 0.5, isopropyl β-d-thiogalactopyranoside was added to 1 mM. Shaking was continued for 5 h. The cells were harvested by centrifugation at 4,000 × g for 10 min, resuspended in 1 mM spermidine Tris hydrochloride/200 mM NaCl/2 mM EDTA, pH 7.5, and frozen in liquid nitrogen. The following steps were performed at 0–4°C. Frozen cells (36 g in 100 ml) were thawed and adjusted to 40 mM Tris⋅HCl, pH 7.6/3.5% sucrose/0.13% Brij-58/1 M NaCl/0.3 mg/ml lysozyme. After incubation for 1 h, the lysate was centrifuged at 90,000 × g for 90 min. To the supernatant, solid ammonium sulfate was added to a saturation of 60% and stirred for 30 min. The precipitate was collected by centrifugation at 90,000 × g for 30 min, dissolved in buffer A [20 mM Tris⋅HCl, pH 7.6/1 mM DTT/0.1 mM EDTA/10% (wt/vol) glycerol] containing 50 mM NaCl, and dialyzed three times against this buffer (Fraction I, 100 ml; Fig. 1, lane d). Fraction I was loaded onto a heparin-Sepharose CL-6B column (2.6 × 15 cm) equilibrated with buffer A containing 50 mM NaCl, and then was washed with 250 ml of this buffer. Proteins were eluted with a 750-ml linear gradient of 50–600 mM NaCl in buffer A. TelN eluted at 430 mM NaCl. Peak fractions were pooled (Fraction II, 175 ml; Fig. 1, lane e). Fraction II was loaded onto a hydroxylapatite Bio-Gel HT column (2.6 × 5 cm) equilibrated with buffer B [20 mM potassium phosphate, pH 6.8/50 mM KCl/1 mM DTT/0.1 mM EDTA/10% (wt/vol) glycerol]. The column was washed with 100 ml of buffer B. Proteins were eluted with a 250-ml linear gradient of 20–500 mM potassium phosphate. TelN eluted at 180 mM phosphate. Peak fractions were pooled (Fraction III, 75 ml; Fig. 1, lane f). Fraction III was diluted with 125 ml of buffer A containing 50 mM NaCl and loaded onto a DEAE-Sephacel column (2.6 × 5 cm) equilibrated with buffer A containing 50 mM NaCl, and then was washed with 60 ml of the same buffer. Proteins were eluted with a 250-ml linear gradient of 50–600 mM NaCl in buffer A. TelN eluted at 160 mM NaCl. Peak fractions were pooled (Fraction IV, 58 ml; Fig. 1, lane g). Fraction IV was diluted with buffer C [50 mM Tris⋅H₃PO₄/1 mM DTT/0.1 mM EDTA/10% (wt/vol) glycerol] containing 50 mM NaCl and loaded onto a phosphocellulose P11 column (1.6 × 9 cm) equilibrated with the same buffer. The column was washed with 80 ml of buffer C containing 50 mM NaCl. Proteins were eluted with a 200-ml linear gradient of 50–600 mM NaCl. TelN eluted at 340 mM NaCl. The peak fraction was concentrated by dialysis against 20% (wt/vol) polyethylene glycol 20,000 in buffer A and than dialyzed against 50% glycerol in buffer A and stored at −20°C (Fraction V, 4.8 ml; Fig. 1, lane h). Under these conditions, the enzymatic activity was stable for at least 1 yr.