Production and recovery of recombinant protease inhibitor alpha(1)-antitrypsin

Human alpha (1)-antitrypsin (AAT) was produced in the recombinant yeast Sac charomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were sim ilar to 55 g .L-1 and 1.23 g .L-1, respectively, in the fed-batch. The maxi mum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L(-1)h( -1), respectively, or substantially greater than the highest productivity v alues reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g .L-1 biomass, 122 min of processing) by cro ss-flow microfiltration and the cells were disrupted by bead milling (3 pas ses of 3 min total retention time). The cell homogenate was treated with al uminum chloride or PBS (pH 7) to aid separation of the cell debris by flocc ulation and sedimentation. The clarified cell homogenate was subjected to a mmonium sulfate fractionation to precipitate the recombinant antitrypsin. T he AAT precipitated at 45-75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25 degreesC), with a zero order deactivation rate of 1.815 x 10(-3) +/- 3.43 x 10(-4) g AAT L(-1)h(-1). (C) 2001 Elsevier Science Inc. A ll rights reserved.