Recombinant yeast technology at the cutting edge robust tools for both designed catalysts and new biologicals
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Recombinant yeast technology at the cutting edge robust tools for both designed catalysts and new biologicals

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Recombinant DNA technology is major DNA-based tool that has gained popular attention in the past decade. Significant advances in the development of new strains and vectors, improved techniques, and ...

Recombinant DNA technology is major DNA-based tool that has gained popular attention in the past decade. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of yeast species have led the recombinant yeast technology a robust tool for both designed catalysts and new biologicals. Yeast combines molecular genetic manipulations and growth characteristics of prokaryotic organisms together with the sub-cellular machinery for performing post-translational protein modifications (O and N- linked glycosylation, disulphide bond formation) and secretion of protein (Intracellularly or extracellularly). A large number of yeast hosts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, etc) are available for heterologous protein expression. The methylotrophic yeast, Pichia species is the most highly developed one among a small group of alternative yeast species chosen for their perceived advantages over S. cerevisiae as a expression host for the generation of recombinant protein of commercial interest. Advantages of the system include the AOX1 promoter (alcohol oxidase) and other alternate promoters (GAP, FLD1, PEX8, and YPT1), with transcription characteristics that are useful for regulating heterologous protein expression.
Auxotrophic mutants (MutS and Mut+) and a new set of biosynthetic markers such as ADE1, ARG4 and URA 3 have been used successfully for better selection of transformed host. Protease deficient hosts and site specific integration of expression vectors into Pichia genome result into high expression of gene of interest. Additional features that are present in certain P. pastoris expression vectors serve as tools for specialized functions. The availability of the expression system as a commercially available kit (Invitrogen) extends the usefulness of system. Several different secretion signal sequences including the native secretion signal or secretion signal sequences from S. cerevisiae such as µ factor prepro peptide causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification. The P. pastoris expression platform is now well developed, as proven by multiple products used in human and veterinary medicine and in industry. A better understanding of secretion signals, glycosylation, and endogenous yeast proteases would be extremely helpful in developing and improving the yeast heterologous expression system.

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Recombinant yeast technology at the cutting edge robust tools for both designed catalysts and new biologicals Presentation Transcript

  • 1. Recombinant Yeast Technology at the CuttingEdge: Robust Tools for both DesignedCatalysts and New BiologicalsPresented by:Navprabhjot KaurPh. D MicrobiologyL-2010-BS-63-D
  • 2. Contents• Recombinant Yeast Technology• Yeast Expression Systems• Problems Associated with Saccharomyces cerevisae• Cloning into Yeast: Pichia pastoris• Features of Pichia Technology• Expression Vectors, Promoters, Selectable markers and Hoststrains• Site-Specific Integration• Post-translational modification• Heterologous proteins expressed in Yeast Expression Systems• Limitations• Conclusions
  • 3. Recombinant DNA Technology
  • 4. Steps in Recombinant DNA Technology
  • 5. Expression Systems5
  • 6. Recombinant Yeast Technology
  • 7. Yeast Expression Systems7
  • 8. Saccharomyces cerevisiae
  • 9. Problems Associated With S. cerevisae
  • 10.  Like Saccharomyces cerevisiae: Easy to manipulate Faster, easier, less expensive than other eukaryoticsystems Advantage over Saccharmoyces cerevisiae: 10-100 fold higher heterologous protein expressionlevels!!10Major Breakthrough in Recombinant Yeast TechnologyCloning into Yeast: Pichia pastorisichia is a methylotrophic yeast(can metabolize MeOH)HCHOO2 H2O2CH3OHAOX
  • 11. Pichia pastoris as an experimental organism
  • 12. Features of Pichia Technology
  • 13. Continued…..Synthetic promoters for fine tuned, both methanol-induced ormethanol-free Gene expression
  • 14. Pichia pastoris as a Methylotrophic Yeast
  • 15. Alcohol Oxidase ProteinsAOX2: Very low level of alcohal oxidase activity
  • 16. Phenotype of aox1 mutants
  • 17. Construction of Expression Strains
  • 18. Selecting a Pichia Expression Vector
  • 19.  EPISOMAL or Plasmid – over expression High copy (20-100 copies per cell) Two origin of replication INTEGRATIVE – introduce gene into yeast chromosome Single copy, aids understanding protein function/role in pathway CENTROMERIC –low copy (YAC)19Cloning into Yeast: Choice of Vector
  • 20. Alternative Promoters
  • 21. PGAP
  • 22. PFLD1 (Glutathione-dependent Formaldehyde Hydrogenase)
  • 23. PPEX8 and PYPT1
  • 24. Selectable markers
  • 25. Host strains
  • 26. Protease-deficient Host Strains
  • 27. P. pastoris host strains
  • 28. Integration of expression vectors into the P. pastoris genome
  • 29. Gene Insertion at AOX I or aoxI:: ARG4His+Mut+(GS115), His+Muts(KM71)
  • 30. Gene Insertion Events at HIS 4
  • 31. Multiple Gene Insertion Events
  • 32. Gene replacement event at AOX I locusHis+Muts(GS115)
  • 33. Transformation
  • 34. Post-translational Modification of Secreted Proteins
  • 35. Intracellular and Secretory Protein Expression
  • 36. Secretion Signal Selection
  • 37. Glycosylation
  • 38. Posttranslational Modifications in comparison to S. cerevisiae
  • 39. Heterologous proteins expressed in P. pastoris
  • 40. Other Yeast Expression Systems
  • 41. Continued….
  • 42. Heterologous Proteins Expressed in Other Yeast Host
  • 43. Limitations
  • 44. Conclusions As a unicellular eukaryote, yeast is quick, easy andinexpensive to genetically manipulate and culture Yeast share many conserved pathways with higher eukaryotesmaking it an excellent platform for studying protein function As well, the wealth of knowledge and set of tools available forYeast species, make it a very powerful genetic tool forstudying protein function High protein yield makes yeast strains useful forpharmaceutical protein production A better understanding of secretion signals, glycosylation, andendogenous yeast proteases would be extremely helpful indeveloping and improving the yeast heterologous expressionsystem.
  • 45. Thank You