Term
| What are the advantages of the yeast expression system? |
|
Definition
1. high yield
2. stable production strains
3. durability
4. cost effective
5. high density growth
6. high productivity
7. suitability for production of isotope-labeled protein
8. rapid growth in chemically defined media
9.product processing similar to mammalian cells
10. can handle -SS- rich proteins
11. can assist protein folding
12. can glycosylate proteins
13. gras organism |
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Term
| Saccharomyces cerevisiae is also known as what? |
|
Definition
| brewers yeast, ale yeast, top-fermenting yeast, bakers yeast |
|
|
Term
| What are the size of the genomic DNA and the number of protein encoding genes in S. Cerevisiae |
|
Definition
Size: 12070 kb
Number of protein encoding genes 6608 |
|
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Term
| What are the industrial applications of non-recombinant yeast and recombinant yeast |
|
Definition
non-recombinant- food poducts (beer, wine, bread, industrial ethanol)
recombinant (insulin, interferons, vaccines) |
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|
Term
| yeast plasmids are ___ vectors |
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Definition
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Term
| TWo major components of S. cerevisiae vectors are a backbone from a bacterial plasmid and a selection marker for yeast. Provide two examples of each |
|
Definition
Backbone (pUC19, pBR)
Selection marker
a. auxotrophic marker (URA3, His2, TRP1)
b. general marker: G418, zeocin |
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|
Term
| The following diagram illustrates the conferred ability module also known as the yeast shuttle vector. provide the names of the following components (1-5) (1-5 in a circle) |
|
Definition
1. Yeast origin of replication
2. Yeast selectable marker
3. origin of transfer
4. Bacterial selectable marker
5. bacterial origin of replication |
|
|
Term
| Describe the difference between strong and weak promoters |
|
Definition
Strong- numerous transcrips and protein molecules
weak -few transcripts and low number of protein molecules |
|
|
Term
| Indicate if the following S. Cerevisae promoters are inducible or constitutive |
|
Definition
Inducible
1. Gal-promoter
2. ADH2-promoter
3. MT-promoter
4. pho5-promoter
constitutive
1. pgk-promoter
2. gapdh-promoter |
|
|
Term
| Sketch the active and inactive state of the Gal-promoter |
|
Definition
|
|
Term
| What are two main systems responsible for protein degradation in S. cerevisase |
|
Definition
1. vacuolar proteases- relased on cell disruption
2. ubiquitin proteasome proteolytic system |
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|
Term
| Vaculolar proteases often released during cell disruption can lead to protein degradation, if inhibitors arent added during downstream processing. Give 4 examples of vacuolar proteases and their corresponding inhibitors |
|
Definition
Protease A (pepstatin)
Protease B (DFP, PMSF)
Carboxylasepeptidase S (EDTA)
Aminopeptidase (EDTA) |
|
|
Term
| What type of protein is degraded by the ubiquitine protease system? |
|
Definition
|
|
Term
| Define the N-end rule of protein degradation by the ubiquitine-proteasome system |
|
Definition
| N-terminal amino acids are recoqnized by multi-ubiquitnylation proteins E3 |
|
|
Term
| Provide 3 features of co-translational processing of the protein N-terminus in S. cerevisae |
|
Definition
1. only in non-secreted proteins with uncleaved N-terminus
2. co-translational cleavage of Met by a MAP
3. N-terminal acetylation following Met cleavage of N-terminal Ala, Ser, Thr
4. N-terminal modification may render proteins less susceptible to aminopeptidase degradation |
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|
Term
| N-termional modification may render proteins less susceptible to ____ degradation |
|
Definition
|
|
Term
| Glycosylation is important for protein _____ and ______ |
|
Definition
|
|
Term
| what are two main types of glycosylation in S. cerevisiae |
|
Definition
Simple O-linked
Complex N- linjked |
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|
Term
| Provide the most common heterologous and homologous signal sequences used for protein secretion in S. cerevisiae |
|
Definition
Heterologous- derived from non-yeast eukaryotic
homologous- alpha-MF |
|
|
Term
| Non conventional yeasts typically yield ____ cell densities |
|
Definition
|
|
Term
| crabtree negative yeast produces very low levels of ethanol under ____ conditions |
|
Definition
|
|
Term
| Provide 2 promoters and thei rcorresponding inducible compound used for high levels of protein expression in Pichia Pastoris and Kluveryomyces lactis |
|
Definition
Aox1(methanol induced)
FLD1 (methanol induced)
Lac 4 (lactose induced
ADH4 ethanol induced |
|
|
Term
| What advantages does P pastoris have over S. cerevisae in terms of heterologous protein expression? |
|
Definition
1. better secretor
2. glycoproteins frequently less N- hypermannosylated
3. grows at high densities in very simple and inexpensive carbon source (methanol) |
|
|
Term
| What are 3 major components of P. Pastoris vectors |
|
Definition
Promoters (AOX1, Gap, FLD1)
secretion signals (alpha MF, Pho1)
marker genes (ADE1, ARg4) |
|
|
Term
| Identify 5 different substrates in which the Crabtree negative yeast can grow on |
|
Definition
glucose
galactose
sucrose
lactose
raffinose
ethanol
glycerol |
|
|
Term
| Kluyveromyces lactis produces a ___ pigment indicating antibiotic activity and has a similar life cycle as ____. This non-convetional yeast is grown on ___ as the sole carbon source in the dairy industry |
|
Definition
pink
s. cerevisiae
lactose |
|
|
Term
| What are the 4 vectors used for protein expression in K. lactis |
|
Definition
1. YEp vectors
2. ARS and centromeric vectors
4. 2 micron vectors |
|
|
Term
| Which is the most common secretion signal used for protein secretion in K. lactis? |
|
Definition
|
|
Term
| Provide 2 examples of proteins expressed in K. lactis along with the corresponding vector and promoter |
|
Definition
human albumin- pkD1, lac4
bovin prochymosin - integ lac 4 |
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