Recombinant Yeast Technology at the CuttingEdge: Robust Tools for both DesignedCatalysts and New BiologicalsPresented by:N...
Contents• Recombinant Yeast Technology• Yeast Expression Systems• Problems Associated with Saccharomyces cerevisae• Clonin...
Recombinant DNA Technology
Steps in Recombinant DNA Technology
Expression Systems5
Recombinant Yeast Technology
Yeast Expression Systems7
Saccharomyces cerevisiae
Problems Associated With S. cerevisae
 Like Saccharomyces cerevisiae: Easy to manipulate Faster, easier, less expensive than other eukaryoticsystems Advanta...
Pichia pastoris as an experimental organism
Features of Pichia Technology
Continued…..Synthetic promoters for fine tuned, both methanol-induced ormethanol-free Gene expression
Pichia pastoris as a Methylotrophic Yeast
Alcohol Oxidase ProteinsAOX2: Very low level of alcohal oxidase activity
Phenotype of aox1 mutants
Construction of Expression Strains
Selecting a Pichia Expression Vector
 EPISOMAL or Plasmid – over expression High copy (20-100 copies per cell) Two origin of replication INTEGRATIVE – intr...
Alternative Promoters
PGAP
PFLD1 (Glutathione-dependent Formaldehyde Hydrogenase)
PPEX8 and PYPT1
Selectable markers
Host strains
Protease-deficient Host Strains
P. pastoris host strains
Integration of expression vectors into the P. pastoris genome
Gene Insertion at AOX I or aoxI:: ARG4His+Mut+(GS115), His+Muts(KM71)
Gene Insertion Events at HIS 4
Multiple Gene Insertion Events
Gene replacement event at AOX I locusHis+Muts(GS115)
Transformation
Post-translational Modification of Secreted Proteins
Intracellular and Secretory Protein Expression
Secretion Signal Selection
Glycosylation
Posttranslational Modifications in comparison to S. cerevisiae
Heterologous proteins expressed in P. pastoris
Other Yeast Expression Systems
Continued….
Heterologous Proteins Expressed in Other Yeast Host
Limitations
Conclusions As a unicellular eukaryote, yeast is quick, easy andinexpensive to genetically manipulate and culture Yeast ...
Thank You
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 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

  1. 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. 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. 3. Recombinant DNA Technology
  4. 4. Steps in Recombinant DNA Technology
  5. 5. Expression Systems5
  6. 6. Recombinant Yeast Technology
  7. 7. Yeast Expression Systems7
  8. 8. Saccharomyces cerevisiae
  9. 9. Problems Associated With S. cerevisae
  10. 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. 11. Pichia pastoris as an experimental organism
  12. 12. Features of Pichia Technology
  13. 13. Continued…..Synthetic promoters for fine tuned, both methanol-induced ormethanol-free Gene expression
  14. 14. Pichia pastoris as a Methylotrophic Yeast
  15. 15. Alcohol Oxidase ProteinsAOX2: Very low level of alcohal oxidase activity
  16. 16. Phenotype of aox1 mutants
  17. 17. Construction of Expression Strains
  18. 18. Selecting a Pichia Expression Vector
  19. 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. 20. Alternative Promoters
  21. 21. PGAP
  22. 22. PFLD1 (Glutathione-dependent Formaldehyde Hydrogenase)
  23. 23. PPEX8 and PYPT1
  24. 24. Selectable markers
  25. 25. Host strains
  26. 26. Protease-deficient Host Strains
  27. 27. P. pastoris host strains
  28. 28. Integration of expression vectors into the P. pastoris genome
  29. 29. Gene Insertion at AOX I or aoxI:: ARG4His+Mut+(GS115), His+Muts(KM71)
  30. 30. Gene Insertion Events at HIS 4
  31. 31. Multiple Gene Insertion Events
  32. 32. Gene replacement event at AOX I locusHis+Muts(GS115)
  33. 33. Transformation
  34. 34. Post-translational Modification of Secreted Proteins
  35. 35. Intracellular and Secretory Protein Expression
  36. 36. Secretion Signal Selection
  37. 37. Glycosylation
  38. 38. Posttranslational Modifications in comparison to S. cerevisiae
  39. 39. Heterologous proteins expressed in P. pastoris
  40. 40. Other Yeast Expression Systems
  41. 41. Continued….
  42. 42. Heterologous Proteins Expressed in Other Yeast Host
  43. 43. Limitations
  44. 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. 45. Thank You

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