TABLE 2.

Purification of 3P proteins from P. pastoris F3P^a

Fraction	Amt of 3P protein (μg)	Yield (%)
Soluble cell lysate	173.8	100
First AS precipitate before dialysis	129.7	74.6
First AS precipitate after dialysis	130.2	74.8
Ni2+-NTA eluate	43.7	25.3
Second AS precipitate after dialysis	23.3	13.4

^aCell lysate was prepared from an induced P. pastoris culture (100 ml). The 3P proteins were precipitated with (NH₄)₂SO₄ (AS) and subjected to Ni²⁺-NTA chromatography. The 3P complex in the imidazole eluate fraction was precipitated by adding (NH₄)₂SO₄, dissolved in storage buffer, and stored at −80°C after dialysis against the storage buffer. A fraction from each step of the purification procedure was subjected to SDS-PAGE, and the gel was analyzed by quantitative Western blotting as shown in Fig. 4. The amounts of 3P proteins were determined by quantitative Western blotting with anti-PB1, anti-PB2, and anti-PA antibodies. RNP was used as a reference control for 3P measurements.