The EtAS gene was ligated into the pYES2/CT vector, in order to purify the EtAS with an Ni2+–nitrilotriacetic acid column. The construct pYES2/CT–EtAS was transformed into S. cerevisiae GIL77. This strain also produced the two minor triterpenes, 3 and 4, in addition to a large quantity of 2. The product ratio was the same as that of pYES2–EtAS. The β-amyrin synthase is expressed as polyhistidine-tag fusion protein (His6, C-terminus), which enabled complete purification in a single step by use of an Ni2+–nitrilotriacetic acid column (Fig. 4A). The yeast cells from a 0.5-L culture were suspended in 30 mL of binding buffer (20 mm Tris/HCl, pH 7.9, containing 10 mm imidazole, 300 mm NaCl, and 0.1% Triton X-100), and then disrupted with glass beads (60 g). The supernatants obtained by centrifugation were pooled after repeated extractions, and then loaded onto the Ni2+–nitrilotriacetic acid resin column. The bound protein was washed with the buffer containing 40 mm imidazole, and finally eluted with the same buffer solution, which also contained 250 mm imidazole. As shown in Fig. 4A (lane 5), the eluted β-amyrin synthase resulted in a single band on 10% SDS/PAGE. The molecular mass estimated from the SDS/PAGE gel mobility was 77–79 kDa (Fig. S3), and the apparent mass was smaller than the calculated one (91.1 kDa). A significant difference between the calculated and apparent molecular masses is frequently encountered; for example, for fission yeast proteins, the observed difference was reported to be at the level of 10–30% . We also found that the molecular mass of the Rv3378c enzyme from Mycobacterium tuberculosis estimated with SDS/PAGE was significantly lower (~ 20%) than the calculated one , but MALDI-TOF MS analyses of the proteolytic digestion fragments showed that the expressed protein was actually of full length . A gel filtration experiment showed that the actual size of this triterpene synthase was 94.8 kDa (Fig. 4B), indicating that EtAS exists as a monomer, as does human lanosterol synthase . In contrast, bovine liver lanosterol synthase was reported to form a dimeric structure . The amount of EtAS expressed by pYES2/CT was estimated by western blotting using an SDS/PAGE gel. The amount of EtAS expressed from 1 L of culture was estimated to be 5.5–6.7 mg. The purified protein was obtained in ~ 50% yield. To measure the CD spectrum of EtAS, Triton X-100 was replaced by Brij35, because Triton X-100 and Tween-80 have benzene rings and double bonds in the molecules that electronically absorb in the UV region and interfere with the CD spectrum, whereas Brij35 has no such functional groups. The bound EtAS on the Ni2+–nitrilotriacetic acid column, described above, was washed with a buffer (80 mL) containing a mixture of 0.01% Brij35 and 80 mm imidazole, and then eluted with a buffer (4 mL) including 250 mm imidazole (Fig. 4A, lane 6). After dialysis, the CD spectrum was measured (Fig. 5), and showed that the enzyme architecture was not altered at temperatures < 30 °C. The enzymatic activity was significantly lower in Brij35 detergent (Fig. 6A). However, substantial molecular ellipticity was observed, suggesting that a higher-order protein structure was maintained in Brij35 solution. Furthermore, the CD spectrum was quite similar to that of SHC, and was superimposable on that measured in a different type of detergent, β-octylglucoside (β-OG) (Fig. S4A).
Figure 4. (A) SDS/PAGE of the expressed EtAS. Lane 1: molecular mass marker. Lane 2: total protein. Lane 3: soluble protein. Lane 4: insoluble protein. Lane 5: purified EtAS [washed with Tris/HCl buffer (20 mm, pH 7.9) including 40 mm imidazole, 300 mm NaCl, and 0.1% Triton X-100, and then eluted with Tris/HCl buffer solution (20 mm, pH 7.9) composed of 250 mm imidazole, 300 mm NaCl, and 0.1% Triton X-100. Lane 6: purified EtAS [washed with Tris buffer (20 mm, pH 7.9) composed of 80 mm imidazole, 300 mm NaCl, and 0.01% Brij35, and then eluted with Tris buffer (20 mm, pH 7.9) containing 250 mm imidazole, 300 mm NaCl, and 0.01% Brij35. Lanes 5 and 6 differ in type of detergent employed. (B) Molecular mass calibration curve based on the retention times of standard proteins obtained by gel-filtration HPLC. The conditions were as follows. Column: TSKgel G3000SWXL. Mobile phase: 50 mm potassium phosphate buffer (pH 7.0), 0.005% Brij35, and 0.2 m KCl. Flow rate: 0.5 mL·min−1, detected at 280 nm. Standard proteins: glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142.0 kDa), enolase (67 kDa), myokinase (32.0 kDa), and cytochrome c (12.4 kDa). The molecular mass of EtAS was determined to be 94.8 kDa).
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Figure 6. (A) Effect of detergent concentrations on the specific activities. The relative activities are plotted. Four different detergents were employed: 0.02–0.75% (w/v) for Triton X-100; 0.02–0.75% (w/v) for Tween-80; 0.2–0.8% (w/v) for β-OG; and 0.04–0.75% (w/v) for Brij35. The (3S)-oxidosqualene concentration used in these experiments was 47 μm. To prepare a solution of oxidosqualene (47 μm) dissolved in β-OG, concentrations > 0.2% were required. (B) Determination of optimal temperature. (C) Determination of optimal pH. The buffer solutions used in the experiment were as follows: 0.1 m Mes buffer for pH 5.5–6.0; 0.1 m potassium phosphate buffer (KPB) for pH 6.5–7.5; and 0.1 m Tris/HCl buffer for pH 8.0–9.0. (D) Determination of incubation times. On the basis of these data, the enzymatic reactions were conducted for 20 min to determine the values of Km and kcat. The (3S)-substrate concentration was 113 μm for (B)–(D), where Triton X-100 (0.05%, w/v) was included in the incubation mixture (highest activity; Fig. 6A). The error bars show the difference between two independent experiments. The specific activity corresponding to 100% relative activity was 352 nmol·min−1·mg−1.
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