Lec 9 - Recombinant Protein Expression Flashcards
Give examples of commercially important proteins that have been produced the DNA recombination technology.
- enzymes - lipases (washing powders)
- antibodies (immunotherapy)
- vaccines - Hep B subunit vaccine
- peptide hormones - HGH, insulin
Give the 5 factors that need to be considered for recombinant protein expression
- How? Which protein expression system will we use (host and vector)
- Make the DNA construct? Vector systems and fusions will we use
- Where will it be expressed?
- Purified? Native protein/affinity tag
- Problems? Protein stability, expression problems?
Give examples of host cells that can be transformed with expression vector
- prokaryotes eg E. coli, Bascillus species
- insect cells - Baculovirus
- mammalian cells
- yeast
Give the pros and cons of using a prokaryotic expression system
Pros :
- easily transformed, quick growth and simple nutrient medium (cheap)
- wide range of commercial vectors available each w/ N/C terminal tags for purification
- express recombinant proteins
Cons :
- post translational modifications may not be completed eg glycosylation
- may secrete -> inclusion bodies therefore insoluble
- problems in removing fusion partner
Describe transcriptional and translational fusions
transcriptional - cloning incoming DNA has no Pro/Ter sites. Vector contains all Pro/Ter/RBS for efficient transcription and translation
translational - incoming DNA fused IN FRAME to other protein (normally used as a tag for purification/ is highly soluble therefore increases solubility of our protein). Your protein needs to be cleaved from tag
Give 5 popular promoters that can be used in vectors
- Plac - need high levels of LacI repressor therefore LacI alleles in vector
- PTrp - trpR repressor w/ Trp present. for expression just starve cells of Trp. not suitable for cells with high Trp content
- Ptac - hybrid promoter. strongly induced by IPTG
- PBAD - araBAD operon. tightly regulated. as soon as arabinose present we get expression
- T7 system - only T7 RNAP recognises the T7 promoter - recognises and elongates along DNA 5x faster than E. coli RNAP
What does pET stand for?
plasmid for Expression of T7 RNAP
Draw a simple diagram highlighting how pET system works
Kelly 2 notes
dont forget IPTG induction to remove LacI repressor
Why would we want to use protease deficient mutants as hosts for transformation into by the vector?
Give examples of proteases that could be mutated
allow the host to minimise the turnover/proteolysis of the gene products
Eg Lon - E. coli protease, ATP dependent and has broad specificity for unfolded/misfolded proteins
OmpT - OM protein that cleaves @ paired basic residues
How can we optimise the transcription of the cloned gene after transformation into the host?
- use strong promoter fusions
- increase gene dosage by using high copy number plasmids
What are some potential problems that arise with optimising transcription?
- protein is too toxic (toxic genes eg membrane proteins) therefore need tight regulation of gene until full growth is reached then expression
- mRNA production is terminated prematurely, mRNA is unstable and is degraded - delete RNAses ?
How can we optimise the translation of the cloned gene after transformation into the host?
- improve SDS (rbs)
- codon usage - some cells may have specific quantities of particular tRNAs/amino acids. rare codons in organisms therefore need to synthesise gene in response to tRNAs that are present in the host cell
- protein may have low solubility/unstable - express @ lower temps/delete the proteases
Give and explain 3 locations where we can express proteins
- cytoplasm - difficult for proteins that require disulphide bonds because reducing conditions. can have thioredoxin mutants that can make the conditions less reducing
- periplasm/secreted - periplasm has oxidising conditions therefore allows correct protein folding BUT can’t get any post-translational modification and limited capacity for secretion
- membrane - only for membrane bound proteins - can become a problem if we get expression of too many MB proteins
Give 3 examples of proteins that are used to tag proteins
- 6X His - bind to Nickel ions on column. elute w/ imidazole
- Glutathione S Transferase - binds to glutathione on Column
- maltose binding protein - binds to amylose on column , elute w/ maltose
Give 2 common problems (and explain in further detail) that can occur w/ heterologous protein expression
- not enough protein produced - too toxic therefore host inserts mutations into promoter regions to reduce expression, mRNA 2ndry structure not stable, preferential codon usage
- protein produced is insoluble/poor folding - need to reduce the time for protein expression - use lower copy number plasmids, use less strong promoters. proteins are more soluble when expressed @ lower temps
