Starting from a gene or cDNA, CBB scientist can clone your gene into any commercially available expression vector or an expression vector provided by the client. A variety of ‘scalable’ host choices are available including E. coli, Saccharomyces cerevisiae, and Pichia pastoris. Following generation of several recombinant lines, the best producing clones will be selected in microtiter plates or shake flasks based on a variety of analysis, such as SDS-PAGE, Western immunoblot, product yield, or enzyme activity. The best clones will then be selected for further optimization and scale-up.
E. coli expression system is the most common expression system. Its physiology and genetics are well-understood. A variety of expression vectors with different tags and fusion partners are available to maximize protein and/or metabolite expression levels. Different host strains with various genetic backgrounds are available to express even complex eukaryotic proteins. E. coli is also amenable to extensive metabolic engineering. CBB expression service in E. coli includes:
- Subcloning of the gene of interest into an E. coli expression vector
- Screening of positive transformants by PCR, restriction digestion of expression plasmids, and DNA sequencing
- Small-scale expression study, optimize expression with different media, induction conditions, and culturing temperature
- Plasmid stability
- Generation of master and working cell banks
- CBB has extensive experience in scaling up inclusion body recovery, solubilization and refolding at large scale (500 liter scale).
Sometimes, proteins are expressed in E. coli as inclusion bodies. CBB may be able to solve this problem by using other expression hosts. CBB also has extensive experience in scaling up inclusion body recovery, solubilization and refolding at 500 L plus scale.
Yeast expression system such as Saccharomyces cerevisiae and Pichia pastoris combine the best features of bacterial and mammalian expression systems. Advantages of yeast expression include:
- Ease of transformation and generation of recombinant clones
- Good expression level
- Relative low cost
- Protein secretion
- Post-translational modification
- Amenable to metabolic engineering
- Well-studied physiology and optimized fermentation process
- Saccharomyces cerevisiae is a GRAS organism
CBB expression service in Saccharomyces cerevisiae and Pichia pastoris includes:
- Subcloning the gene of interest into a Saccharomyces cerevisiae and/or Pichia pastoris expression vector
- Screening of positive transformants by PCR, restriction digestion of expression plasmids, and DNA sequencing and confirmation of sequence fidelity
- Confirmation of integration of expression vector into Pichia genome by PCR and Southern blot; screening of transformants for gene copy number
- Small-scale expression study, optimization of expression under various, growth and induction conditions
- Scale-up expression levels at 2-1000 L
CBB has many years of experience in expressing different proteins in yeast. Please contact us to discuss detailed specification of your project.