Pichia pastoris offers many of the advantages of other eukaryotic systems, such as disulfide bond formation, proteolytic processing, protein folding, and glycosylation. It grows in relatively simple medium, lacks endotoxin, and genetic manipulations are straightforward. Pichia pastoris also has an efficient secretory pathway, so proteins can be produced in conditioned medium, simplifying downstream processing.
1. Target gene(s) are cloned into a pJ expression plasmid (Biogrammatics) with N or C-terminal His and/or Strep tags and an n-terminal alpha mating factor secretory signal peptide and up to 100 ug of plasmid DNA is produced.
2. The DNA is introduced into a yeast strain and clones are selected for stable integration of the target DNA on medium containing antibiotic.
3. Small-scale expression analysis of the target protein in two or more clones is then performed over a five day time course, with samples of cells and conditioned medium harvested each day. Samples of conditioned medium are analyzed by PAGE and Western blot and the clone and time point exhibiting the highest expression of secreted protein is selected for affinity pulldown analysis. Affinity pulldowns are conducted from conditioned medium and analyzed by PAGE and Western blot.
4. 1 liter expression in shake flasks is performed and harvested at the expression conditions identified in the small scale expression analysis. The protein is purified by affinity chromatography using nickel resin or tandem affinity chromatography using nickel and streptactin resins, with or without polishing by SEC. Elution fractions are analyzed by PAGE & Western blot and pooled based on purity metrics established with the client.
1) Catalog No.Custom includes vector construction, plasmid production, and strain creation.
2) Catalog No.Custom includes small-scale expression analysis.
3) Catalog No.Custom includes 1L expression, and 1 step affinity purification.
4) Catalog No.Custom includes 1L expression, and tandem affinity purification (>90% purity is typically achieved but is not guaranteed)
5) Catalog No.Custom includes 1L expression, tandem affinity purification, and an SEC polishing step.
Please note: production may subsequently be scaled up to 10 liters or more to produce protein quantities needed by clients.
Options available: low endotoxin processing, affinity tag removal by protease cleavage, protein titer quantification (HPLC), analytical protein characterization including a SEC-HPLC, C-IEF, thermal stability analysis, mass-spec, protein formulation optimization, and expression in other yeast systems.
DNA encoding target protein
1. Purified protein
2. Study report
3. Certificate of Analysis