Recombinant Engineering in Protein Science Flashcards
Why is recombinant engineering useful?
- can add purification tags
- Prepare plasmid vectors for protein extraction
- express small part of a larger protein complex
- make mutant proteins
Why use recombinant proteins?
- Not all proteins are abundant
- Not all organisms can be cultured or are safe (e.g. extremophiles)
What are the difficulties associated with recombinant expression in host cells?
- localisation
- toxicity
- folding characteristics
- post-translational modifications
What can fusion proteins/tags be used for?
- Purification
- Enhancing expression levels
- Protein detection
How can fusion proteins/tags be useful for protein detection?
You can add antibody epitopes, fluorescent proteins, enzymes
Why is His-tagging useful?
- Multiple His not found in nature
- High purity in one step
- Interacts with Nickel resin
- Universal technique
- Easy, cheap, reliable
Why are synthetic genes useful?
- No need to obtain genomic DNA
- No need for cloning by PCR
- Gene sequence optimized for expression in recombinant host
- Can make artificial proteins
How is site directed mutagenesis usually done?
Using mutagenic PCR primers
Why is site directed mutagensis useful?
- Powerful way of understanding and engineering proteins
- Rational process which allows for direct testing of amino acid functions
What are the problems with site-directed mutagenesis?
- Changing a single amino acid can have dramatic effects
- Helps to have crystal structures
- Results can be tricky to interpret
Why should you choose a mutation that deletes part of the side chain or causes isosteric change?
- Any increase in sidechain volume can distort structure
- Small cavities caused by deletions are tolerated
Why is it important to avoid creating buried unpaired charges?
Solution energies of ions are very high and unpaired charged groups must be solvated
Why is it important to delete the minimum number of interactions?
It is hard enough to interpret the loss of 1 interaction, let alone multiple
Why is it important to not introduce any new functional groups?
This can lead to new interactions that can distort protein structure
Why should you mutate to alanine if in doubt?
It is a deletion mutation to an inert side chain
