EVALUATION OF DIFFERENT PREPARATION PARAMETERS FOR THE PRODUCTION ANDCRYOPRESERVATION OF SEED CULTURES WITH RECOMBINANT SACCHAROMYCES-CEREVISIAE

In this article we describe the intensive investigation of defined phy sicochemical parameters for their influence on the preparation, storag e stability in liquid nitrogen, and the post-thaw productivity of a re combinant strain of Saccharomyces cerevisiae expressing the human fact or XIIIa protein, Preparation of industrially sized seed cultures and their storage stability were monitored over more than a 1-year period. Major parameters recorded before and after thawing were number of col ony-forming units on agar slant media, plasmid retention, and expressi on capacity of the recombinant protein. The viability of the cells aft er reconstitution of the frozen batches was found to improve significa ntly if a certain combination of prethaw growth conditions was applied and amino acids were included in the cryoprotectant media. In additio n, the influence of different concentrations of glycerol as a cryoprot ectant was investigated and the specific freezing conditions which imp roved the viability were identified. Even without complex freezing dev ices for the controlled freezing of cultures, viability rates of 85% a nd higher were obtained, enabling consistent production of the recombi nant protein. (C) 1995 Academic Press, Inc.