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Flashcards in Lecture 5 Deck (45):

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)


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

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
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?



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?

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

1. Make construuct in Ecoli
2. linearise
3. transform into pp
4. Homo recomb occurs between regions of AOX (at either end of segment)
5. select for his+ marker on integrated dna


Why are large parts of the alc ox gene included up and downstream of gofi?

Cos plasmid not very stable in euk so need strong regulation.


Is the homologous recomb event common?

Nope. Rare infact.


List 3 proteins which have acheived high yields through the pp expression vector system

tumour necrosis factor
rat erythropoetin
alpha amylase


Name an alternative to the PP expression vector system (one that would be better for humans)

Baculovirus in insect cells


What is a baculovirus?

virus that infects invects invertebrates inc many insect cell types


what is the baculovirus expression system capable of?

post translational modifications
remove introns


Describe the life cycle of the bascilovirus

infecticve, rod shaped capsids that can bud infect cell.
Hijak 'cription and 'lation
nuclear occlusion bodies, called polyhedra, ie the new baby viruses bud out
These are embedded in a matrix of polyhedrin protein
This converts the virus to it's long term survival form
now wait till eaten by another insect .


What happens when survival form (virions) of baculovirus is eaten by another insect

pH change causes this protein to degrade
virions released and form viruses.


Why is the polyhedrin protein so important to basculovirus' ability to be a good expression system?

because very regulated so has very strong promotor


How is the gene of interest actually inserted into the viral genome?

via the Autographa californica nuclear polyhedrosis virus first - type of baculovirus


Describe the process of preparing the recombinant baculovirus

Prepared in ecoli using a bacimd
Clone gene of interest into donor plasmid, so DNA is downstream of polyhedrin promotor and followed by terminator sequence
Introduce this into Ecoli conatining bacmid
Recombination occurs between att (attachment) regions


What is a bacmid and what does it contain?

genetically altered AcMNPV
ecoli origin
selectable marker
Lacz (surrounding attachment site)


What is the Lacz in place of in the bacmid

the polyhedrin gene


What is the point of the Lacz?

If donor gene correctly inserted Lacz will be disrupted . If it is not disrupted, a peptide which is visibly blue is formed, so you know if you've been successful.


Now have a recombinant bacmid (if got amp resistance and white peptide.) Where is it now transfected?

Insect cells


Which cells and cell lines are used to accept the recombinant bacmid?

Spodoptera frugiperda. Yes worms.
Cell line sf9 and sf21


Can insect cells be used in continuos cutlure when infected with virus?

NO - cells die at the end, but you still get a high level of recombinant protein.


What have the womrs cells been modified to allow?

co expression of 2 genes at the same time using a second promotor called a p10 - allows formation of complexes.


can some baculoviruses inject mamaallian cell lines?



Why are insect cells still not ideal for expression of proteins used by humans?

different glycosylation pathways. Gareth lurves glycozz.


How could the glycosylation issue be got around?
Once you've got the answer what's an example of when that's been done?

co express human modification enzymes at same time as recombinant protein
human tissue plasminogen activator is correctly glycosylated hallelujah.