Lecture 5 Flashcards
Recombinant protein expression in Eukaryotes (NOT E.coli like lect 4)
state 3 problems with recomb expression in E.coli
not able to produce right post translational modifications
Recombinant proteins can contain contaminent proteins which generate immune response in next host
euk proteins often insoluble in ecoli
Why are euk proteins insoluble in ecoli
cos correct chaperones not present
list 4 post translational modifications
glycosylation (N or O linked sugars)
acetylation
palmytilation
phosphorylation
Name 3 possible roles of post translational modifications
important for mode of action of therapeutics via target site (which might recognise glycan)
role in folding
role in final conformation
In terms of glycosylation, what makes humans unique?
a cyclic acid attached
Name a fungal eukaryotic alternative to ecoli
Saccharomyces cerevisiae
List the positives of S.cerevisiae as expression vector
cheap easy to grow genetically ameanable 6000 genes (2000 more than ecoli) contains plasmids strong natural yeast promotors available
Give 4 examples of yeast promotors
glyceraldehyde phosphate dehydrogenase
phosphoglycerate kinase
galactokinase
acid phosphotase
What is the purpose of using a shuttle vector
Purpose - enables cloning to be done in ecoli, from which it is easy to prepare lots of plasmids. Then moved into yeast
List the components required for a s. cerevisiae shuttle expression vector, trying to give specific examples
2 oofr - 1 for yeast plasmid, 1 for ecoli.
Promotor region - e.g. GAPD
Terminator equivilent of promoter - e.g. GAPDt
cDNA copy of gene you want
antibiotic resisrnace gene marker
nutritional deficiency marker
Why does the shuttle vector need a nutritional marker
because ab have no effect on eukaryotes which yeast is
What is essential if glycosylation is to occur
for the recomb prot to travel through ER
Explain the process of causing a recomb protein to glycosylate in S.cere.
Fuse signal peptide from yeast mating type factor alpha1 gene
Signal Recognition Particle targets signal peptide to translocon on ER membrane
Signal pep cleaved at lys-arg
Proteins folded and modified in ER lumen
Preassembled glycan added to Asn at Asn-X-Ser/Thr
Glycan further processed in golgi, then secreted in vesicle
3 problems with S.cere as expression system
often plasmid loss in fermenters cos such large scale cultures
hyperglycosylation - addition of 100 manise residues in each N linked side chain.
If recomb protein not normally glycosylated and contains Asn-X-Ser/Thr motif it will be glycosylated
What are the possible results of hyperglycosylation
alter protein propperties and activities
Immunogenecity - recognised as foreign and cleared, reducing half life.
Name a better alternative to S. cere (and Ecoli)
Pichia Pastoris
What type of yeast is PP and what does this mean?
methylotrophic - can grow on methanol
gives X fold greater levels of expression of intracellular and secreted proteins that SC. X is?
10-100
2 reasons why better than SC
MM good for secreted proteins Because doesn’t secrete many of its own (but will if given right signal)
doesn’t hyperglycosylate
How is PP able to use methanol as a sole c soure? And why is this beneficial to its role as expression vector?
Because it expresses alcohol oxidase
and has a very strong promoter for it which can be exploited
What regions of alcohol oxidase gene can be used in expression vector
promotor (AOX1p)
terminator and polyadenylation signal region (AOX1t)
What is the alc ox gene repressed and induced by
repressed by glucose
induced by methanol
What exactly does the expression vector contain for use in PP?
AOX1p-cDNA-AOX1t HIS4 - a selectable marker 3'-AOX1 region homologous to DNA just downstream of end of AOX1. ori for ecoli AB selectable marker for Ecoli
Describe the integration of the PP vector onto chromosome
- Make construuct in Ecoli
- linearise
- transform into pp
- Homo recomb occurs between regions of AOX (at either end of segment)
- select for his+ marker on integrated dna
