A rapid method for the purification of Neurospora crassa tyrosinase (EC 1.14.18.1) has been developed using an FPLC system that yields sufficient pure protein for protein crystals to be grown.
Neurospora crassa wild type (74A) was grown at 30 C in shake culture in full-strength Vogel's medium, 2% sucrose, from a large conidial inoculum according to the procedure by Lerch (1987 Meth. Enzymol. 142:165-169). Tyrosinase was induced by transferring the mycelia into fresh half-strength Vogel's medium, 0.5% sucrose, containing cycloheximide to a final concentration of 1.5 uM (Horowitz et al. 1970 J. Biol. Chem. 245: 2784-2788). The mycelia were harvested by filtration under low vacuum, dried with paper towels, and ground in sand with a pestle and mortar in the presence of 0.1 M Tris-HCl buffer, pH 8.0 and 2 mM sodium benzoate (buffer A). After stirring for one hour, the crude extract was centrifuged and the supernatant collected. The supernatant was then applied to a Q-Sepharose column (2.6 x 28 cm) pre-equilibrated with buffer A. After washing the column with two column volumes of buffer A, tyrosinase activity was eluted with a linear gradient (0-0.75 M) of NaCl over 600 ml in buffer A. A wide peak of protein showing tyrosinase activity was eluted between 0.18 and 0.54 M NaCl. The pooled activities from the tyrosinase peak were desalted on a Sephadex G-15 column (5.0 x 16 cm), pre-equilibrated in 50 mM sodium citrate buffer pH 5,0 containing 2 mM sodium benzoate (buffer B). Active fractions were loaded onto a Mono-S HR5/5 column pre-equilibrated in buffer B. Activity was eluted with a linear gradient (0-0.3 M) of NaCl over 12 ml in buffer B. Protein containing tyrosinase activity eluted as a single peak at 0.2 M NaCl. The peak fractions appear as a single band on SDS-PAGE. Samples of each of the steps during the purification are presented in Figure 1.
The purified protein was concentrated to 15 mg/ml using a Centricon 10 spun at 5,000 x g for 1 h. Protein was crystallized by the hanging drop method (Ducroix and Giege, Methods of Crystallization, pp. 73-98 in Crystallization of Nucleic Acids and Proteins: A Practical Approach. A. Ducroix and R. Giege (eds.), IRL Press, Oxford, UK). The reservoir contained 4-6% w/v polyethylene glycol MW 12,000 in 0.3 M MOPS/ethanolamine buffer, pH 7.2, at 18 C, in the presence of 50 mM thiourea as an inhibitor of tyrosinase activity. After three days tetragonal needles 0.03 x 0.03 x 1.5 mm were formed, as shown in Figure 2. Improving the size of the crystals to enable X-ray structure analysis of tyrosinase is the subject of current work.
Figure 1. SDS-PAGE gel stained with Coomasie brilliant blue R250 showing the purification of tyrosinase.
(A) Prestained protein molecular weight standards, (B) Cell culture supernatant, (C) Crude extract of
Neurospora, (D) Post-cell debris supernatant, (E) Tyrosinase from Q-Sepharose, (F) Tyrosinase from G-15,
(G) Tyrosinase from Mono S HR5/5.
Figure 2. Long tetragonal needles of Neurospora tyrosinase. The square cross-section of the needle can be
clearly seen.