Expression of tetanus toxin fragment C.
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.
Medicines Research Centre, Glaxo-Wellcome, Stevenage, UK.
J
J
Clare
K
K
Sreekrishna
M
M
Romanos
D000429:Alcohol Oxidoreductases / Q000235:genetics; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods; D003017:Clostridium tetani / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005285:Fermentation; D005822:Genetic Vectors; D007202:Indicators and Reagents; D009466:Neuromuscular Blocking Agents; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics / Q000254:growth & development; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013744:Tetanus Toxin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
D007202:Indicators and Reagents; D009466:Neuromuscular Blocking Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase