Protein production Flashcards
What are fusion tags?
independent of host organism
easy protein identification & purification
improve stability & folding
can be added N/C -terminal & in-frame
If at N-terminus, remove ATG
if at C-terminus, remove stop codon
How can you purify tagged proteins?
Affinity Chromatography
affinity ligand associated with resin
contaminating proteins wash off
wash off protein using solution of appropriate ligand
What is the affinity column used for Glutathione-S-transferase?
Glutathione
What is the affinity column used for Maltose binding protein?
Amylose (from starch)
What is the affinity column for Hexa-histidine tag?
Metals (Nickel or cobalt)
What is the affinity column for StrepTag II?
StrepTactin - engineered bacterial protein
how can fusion tags be associated with protease cleavage sites to allow removal?
Recognition sites
What are advantages of E.coli as a host for recombinant expression?
simple & rapid
easy to transform
well characterised
range of vectors
What are the disadvantages of E.coli as host for recombinant protein expression?
requires cDNA - E.coli doesn’t recognise introns
lacks post-translational processing
protein stability issues
What is the T7 promoter system?
2 key elements - incorporated into genome. expression of T7 polymerase is controlled by the lac UV5 promoter
What are some codon usage problems?
E.coli tRNA can become depleted
choosing a host strain with additional tRNA genes for rare codons can improve translation
What does glucose depletion result in small quantities of?
lac permease - allows small amounts of lactose in
converted to allolactose - induces system, prevents lac repressor binding
What are advantages of expression in fungal cells?
flexibility over copy number of the plasmid vector
some eukaryotic post-translational modifications e.g. proteolytic processing
deletions of genes for homologous proteins = functional assays in vivo by complementation
cheap
easy to grow
high yields
produce ion channel protein for structural biology
What are the 2 approaches to driving expression far Sacchromyces cerevisiae?
Autonomous replication
Integrative
What facts bout expression in Pichia pastoris?
AOX1 promoter - drives pacha cell production of the AOX1 enzyme
yield up to 30% of total cell protein
What are problems with expression in Pichia pastoris?
does not support episcopal DNA - requires chromosomal integration
Low transformation efficiency
Problem with hyperglycosylation
cells need to be aerated - often need for oxygen
How does the AOX1 promoter in Pichia pastoris work?
promoter is repressed by glucose
induced by methanol
with from Glc to MeOH = expression
What are the 2 vector integration strategies for Pichia pastoris?
integration of AOX1 - alcohol oxidase produced, cells can metabolise methanol
Replacement of AOX1 - chromosomal AOX1 replaced, no AOX1 enzyme, cannot metabolise methanol
How are pichia grown in bioreactors?
agitator
thick cultures
feed in for air - high dissolved oxygen for alcohol oxidase activity
What are the 2 ways higher eukaryotic cells are grown by tissue culture?
cell monolayers in flasks
suspension cultures in spinner flasks
What are the steps for an expression system in insect cells?
insert cDNA for target protein in transfer vector
transfect insect cells - produce recombinant virus containing cDNA
infect cell culture at optimal pointing growth cycle
test cells for expression of protein
What are key features of the insect cell expression system?
viral polyhedrin promoter
polyhedron promoter is active in late stage infection
Baculovirus can be used with Sf9 and Sf21
How is a recombinant baculovirus genome constructed?
transfer vector encoding protein COMBINED with baculovirus DNA
done by DOUBLE CROSS-OVER RECOMBINATION or TRANSPOSON-MEDIATED RECOMBINATION
How does double cross over recombination occur? (baculovirus)
POI is downstream of polyhedrin promoter
vector contains orf603 and orf1629
double recombination between 2 odd sequences - incorporate EXPRESSION CASSETTE WITH POLYHEDRIN PROMOTER