Upc 900

For AviPure purification, 5 g biomass (pellets) from the previously mini-pilot scale fermentation was re-suspended in 40 mL sonication/Wash buffer (50 mM Phosphate Buffer pH 7.5, 300 mM NaCl, 10 mM Imidazole) and 350 µL protease inhibitor cocktail mix. Direct sonication was performed for 45 min using amplitude of 90 % and 0.6 cycles; with the beaker containing cell suspension placed in ice/ice cold water and was continuously stirred to keep cool. After sonication, the lysate was centrifuged for 1 h at 16,000 rpm using a Beckman Coulot Avanti J-E centrifuge to get rid of cell debris. IMAC was used to purify the target protein and a 5 mL Ni–NTA column volume was chosen, equilibrated with sonication/wash buffer before loading. After centrifugation, the final 40 mL supernatant was loaded on the column. UPC–900 Amersham Biosciences AKTA FPLC system was employed for the purification. The elution buffer used was the same as the sonication/wash buffer, but contained a higher imidazole concentration (250 mM). The obtained protein was analyzed by SDS-PAGE following the method of Laemmli.

Before separation, YHL was filtered using 0.45-μm filters. The separation of each component was made on an AKTA purifier (Amersham Pharmacia Biotech; Uppsala, Sweden) equipped with a pump (P-920), UV monitor (UPC-900), a valve (INV-907), a mixer (M-925), and a fraction collector (Frac-920). The separation was performed in a 5 mL HiTrap desalting size exclusion column at room temperature with 3 mL/min of sodium citrate buffer (pH 4.80 and 50 mM) as mobile phase. The injection volume was 0.5 mL and injections were carried out 14 times to separate 6.29 g of YHL (7.5 g YH contains 6.29 g YHL). When there is no peak, Frac-920 collects 1 mL fraction in each tube (Figure 1). When there is a peak, the peak will be collected as one large fraction or in several tubes each collecting at most 1 mL fraction (Figure 1). A 0.33 mL of Celluclast 1.5 L was added to a 10-mL volumetric flask and diluted with sodium citrate buffer to obtain 40 g/L solution for component separation.

One milligram (1 mg) of commercial horse F(ab’)₂ anti-botulinum AB (bivalent), anti-diphteric, antitetanic or anti-rabies immunoglobulins were subjected to molecular exclusion chromatography on a Superose 12 HR 10/30 column (Amersham Pharmacia Biotech AB, Sweden), equilibrated and eluted with ammonium acetate 50 mM, pH 7.4. Samples were run at a 24 mL/h flow rate, and their protein content was monitored by recording the absorbance at 280 nm in a UPC-900 Amersham Pharmacia Biotech.