ABSTRACT
We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.
Subject(s)
Biotechnology/methods , Genetic Techniques , Pichia/metabolism , Proinsulin/genetics , Proinsulin/metabolism , Alcohol Oxidoreductases/genetics , Amino Acid Sequence , Cloning, Molecular , Disulfides/chemistry , Electrophoresis, Polyacrylamide Gel , Fermentation , Gene Dosage , Genetic Vectors , Humans , Kinetics , Mass Spectrometry , Molecular Sequence Data , Peptide Mapping , Peptides/chemistry , Plasmids/metabolism , Promoter Regions, Genetic , Saccharomyces cerevisiae/metabolism , Serine Endopeptidases/metabolism , Time FactorsABSTRACT
Increased expression of recombinant mini-proinsulin in Pichia pastoris in 2.5 l bioreactors was achieved by increasing the cultivation pH from 5.1 to 6.3, by decreasing the temperature from 28 to 22 degrees C, and by periodical addition of ammonium sulfate and EDTA to the culture broth. Using this procedure, mini-proinsulin reached nearly 0.3 g l(-1) in the culture supernatant after 160 h of growth.
Subject(s)
Bioreactors/microbiology , Cell Culture Techniques/methods , Nitrogen/metabolism , Pichia/growth & development , Pichia/metabolism , Proinsulin/biosynthesis , Ammonium Sulfate/pharmacology , Cell Proliferation/drug effects , Edetic Acid/pharmacology , Hydrogen-Ion Concentration , Pichia/drug effects , Pichia/genetics , Proinsulin/genetics , Recombinant Proteins/biosynthesis , TemperatureABSTRACT
The immunogenicity of sticholysin II (St II), a pore-forming polypeptide from the sea anemone Stichodactyla helianthus, was studied in rabbits using two adjuvants, Freund's and aluminium hydroxide. High titres of antibodies were raised against St II with Freund's adjuvant (FA). The structural homology between sticholysins I and II was also revealed by cross-reactivity assays. Since the oil constituent of FA neutralized the St II haemolytic activity, immunizations with St II-Freund's emulsions were carried out with the inactivated cytolysin. Purified anti-St II IgG also neutralized the St II haemolytic activity.