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Methods Mol Biol. 1998;103:193-208.

Expression of tetanus toxin fragment C.

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  • 1Medicines Research Centre, Glaxo-Wellcome, Stevenage, UK.


1. The yield of fragment C was only modestly affected by Mut phenotype, and the site and type of integration event. 2. Fragment C accumulation was closely correlated with gene dosage and maximal expression levels required high gene copy number. 3. Yields were greatly increased in controlled fermenters, compared to shake-flasks, owing to the high cell density achieved and to an increased efficiency of induction (2.5- to 10-fold). 4. In fermenter inductions of a 14-copy strain, fragment C accumulated to 27% of total protein, giving an estimated yield of 12 g/L. 5. Considerable clonal variation in the level of expression occurred with transplacement transformants, and this was owing to a diversity of different integration events and to differences in gene copy number. 6. These multicopy transplacement events occur by in vivo circularization of transforming DNA fragments followed by repeated single-crossover integration. This is presumably a general phenomenon, such that it should be possible to obtain multicopy integrants from all P. pastoris transplacement transformations.

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