recombinant yeast technology at the cutting edge robust tools for both designed catalysts and new...
DESCRIPTION
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.TRANSCRIPT
Recombinant Yeast Technology at the Cutting Edge: Robust Tools for both Designed Catalysts
and New Biologicals
Presented by:
Navprabhjot Kaur
Ph. D Microbiology
L-2010-BS-63-D
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 Host strains
• Site-Specific Integration
• Post-translational modification
• Heterologous proteins expressed in Yeast Expression Systems
• Limitations
• Conclusions
Recombinant DNA Technology
Steps in Recombinant DNA Technology
Expression Systems
5
Recombinant Yeast Technology
Yeast Expression Systems
7
Saccharomyces cerevisiae
Problems Associated With S. cerevisae
Like Saccharomyces cerevisiae: Easy to manipulate Faster, easier, less expensive than other eukaryotic
systems Advantage over Saccharmoyces cerevisiae:
10-100 fold higher heterologous protein expression levels!!
10
Major Breakthrough in Recombinant Yeast TechnologyCloning into Yeast: Pichia pastoris
Pichia is a methylotrophic yeast(can metabolize MeOH)
HCHO
O2 H2O2
CH3OH
AOX
Pichia pastoris as an experimental organism
Features of Pichia Technology
Continued…..
Synthetic promoters for fine tuned, both methanol-induced or methanol-free Gene expression
Pichia pastoris as a Methylotrophic Yeast
Alcohol Oxidase Proteins
AOX2: 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 – introduce gene into yeast chromosome Single copy, aids understanding protein
function/ role in pathway
CENTROMERIC –low copy (YAC)
19
Cloning into Yeast: Choice of Vector
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:: ARG4
His+ Mut+ (GS115), His+ Muts (KM71)
Gene Insertion Events at HIS 4
Multiple Gene Insertion Events
Gene replacement event at AOX I locus
His+ 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 and inexpensive to genetically manipulate and culture
Yeast share many conserved pathways with higher eukaryotes making it an excellent platform for studying protein function
As well, the wealth of knowledge and set of tools available for Yeast species, make it a very powerful genetic tool for studying protein function
High protein yield makes yeast strains useful for pharmaceutical protein production
A better understanding of secretion signals, glycosylation, and endogenous yeast proteases would be extremely helpful in developing and improving the yeast heterologous expression system.
Thank You