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Protein biosynthesis was induced by methanol for 1 or 3 days [13] and [16]. The induced atrial natriuretic peptides were then precipitated by centrifugation (2000g, 10 min) and stored at ?80 °C. Cells were suspended in 20 mM KHPO4 (pH 7.4)/0.5 M NaCl/1 mM GSH/10 mM imidazole/0.04% Tween 20/5% glycerol and with protease inhibitors (Roche) and disrupted with glass beads [16]. After centrifugation (30,000g, 10 min, +4 °C), the supernatant was stored at ?80 °C, and an aliquot (0.5–2 ml) was used for assay of enzyme activity. pPICZB-LDS yielded several transformants with identical enzyme activities, and one was chosen for further studies. Expression of mutants of 7,8-LDS, which were inactive, was confirmed by Western blot analysis, as described below. The expression experiments were repeated several times with the same results.
Pichia cells transformed with pPICZB-LDS were also grown in basal salt medium in an FE2 bioreactor at 20 °C with 20% dissolved oxygen and pH 5.0, controlled by addition of NH3 and H3PO4 [17]. Protein biosynthesis was induced by 0.5% methanol. The culture reached an OD600 of 600 on day 5. The cells were recovered by centrifugation, stored at ?80 °C, processed as above, and used for studies of catalytic properties of recombinant LDS.