Biotechnology 6 Flashcards
Why do we need recombinant proteins?
Mass production
Protein manipulation
What is a therapeutic use of recombinant proteins?
Insulin
tPN
EPO
What are 3 important consideration when choosing expression systems?
Size of target protein
Solubility of target proteins
Post translational modifications
Why is E Coli used as a common host cell?
Simple Cheap Fast grow High yield Genetics
What components can be optimised with E coli?
The E coli strain
The plasmid vector
The coding cDNA sequence inserted
What are the essential components of expression plasmid design?
Promoter `ribosome binding sequence Cloning site Transcription terminator Selection gene Replication origin
What are the optional components of expression plasmid design?
N terminal sequence Signal peptide for secretion Fusion tag for purification PolyA signal Chaperone encoding gene
What is lac promoter used for?
Inducible promoter
What does a lac promoter do?
Regulates transcription of the lac operon
What happen sand lactose is absent (the lac promoter)?
Repressor is active and lac operon is off
What can the lac promoter be used for?
Improve protein yield
Define inducible promoter
Allow the expression of the recombinant protein to be switched on only when the bacterial culture has been established
Why are inducible promoters desirable?
Recombinant proteins are often toxic for bacteria and cause slow growth which doesn’t occur with inducible promoters
Why do codons need to be optimised?
There are differences between codon usage in E coli and humans
therefore corresponding tRNAs are not found in the same proportion
Identify 2 means of optimising codon usage
Replace human favoured codons for bacterial favoured ones by chemical cDNA synthesis and site-directed mutagenesis
Use modified E. coli strains overexpressing rare tRNAs
What are benefits of incorporating N terminal fusions?
Can improve translation rate
Can increase protein stability
Can facilitate purification
Define fusion tag
A specific type of C or N terminal fusion designed to facilitate subsequent purification of recombinant proteins
When can a N terminal fusion be removed?
After purification if it linked to the rest of the protein by a suitable protease target sequence
What is a polyhistidine tag?
For affinity purification on an immobilised nickel chromatography column
Give 2 examples of fusion tags
MBP
GST
What is an advantage of inducing recombinant protein secretion?
E. coli cytosol is not favourable to foreign protein folding whereas its periplasm is
Large amounts of intracellular proteins can form aggregates
To improve protein solubility and facilitate harvesting
What are the benefits of adding N terminal signal peptide?
In periplasm host proteases can cleave the signal peptide
Periplasmic host enzymes also assist protein folding
Osmotic shock is sufficient to release recombinant proteins (no cell lysis)
It can improve protein stability and solubility
Why is recombinant human insulin used?
Cheap
Limitless quantities
Non-immunogen
How is recombinant human insulin produced?
A & B chains fused to β-galactosidase for stability
Transformed separately in 2 different E. coli
Affinity-purified with column/beads bound to β-galactosidase ligand
Cleavage of β-galactosidase with cyanogen bromide
A & B mixed, refolded, cysteine oxidized to form S-S bonds
What are 3 reasons for protein engineering?
Improve protein stability
Modify protein kinetics
Create something new
What are 2 requirements of protein engineering?
Detailed knowledge of protein
Site-directed mutagenesis or chemical gene synthesis
What are 3 examples of protein engineering?
Fluorescent proteins
tPA
Subtilisin from Bacillus subtilis
Name 4 examples of natural microbial compounds
Flavouring compounds
Organic solvents and acids
Secondary metabolites
Polysaccharides
Define bioremediation
Reclaiming or cleaning up contaminated sites using microorganisms to remove or degrade toxic wastes.
Give 2 applications of bioremediation
Oil spills
Chemical degradation in soils
