Protein Engineering Flashcards
What is protein engineering?
Protein engineering involves developing proteins to improve or change their characteristics e.g. better for manufacturing, stability, substrate specificity etc
What is the process of protein engineering?
- A library is created that has sequence diversity.
- Mutants with improvements are screened or selected.
- This is repeated using successful mutants as a scaffold.
What is rational design?
Rational design uses knowledge about the protein to determine mutants that should be produced. This reduces the library size to around 1-10 mutants.The knowledge could include annotated structure, mechanism, specific residues or regions that affect a certain property, knowledge of specific amino acids.
Describe 2 methods of rational design.
Site-directed mutagenesis - This involves choosing a residue or small region that you wish to change. Primers with different sequences are used to produce this library. e.g. adding a cysteine residue so a fluorophore could be attached close to the binding site so that binding could be compared.
Scanning Mutagenesis - This involves changing each residue in the sequence to the same amino acid (often alanine) to determine residues that are important for function.
What is combinatorial design?
This mimics evolution by using a known DNA template and making random changes to it. This requires multiple rounds and produces a large library size. This is best for properties that occur across the whole protein e.g. stability, as you are unlikely to find an improvement to specific regions due to it being random. The process used is called random mutagenesis.
Describe 2 methods of random mutagenesis.
Error-prone PCR - uses polymerase with no proofreading capabilities and then additional features to increase the mutation frequency e.g. excess Mg2+ /Mn2+, unequal dNTPs, more cycles, more enzymes.
In-vivo mutagenesis - This uses living cells and applies a mutagen to cause mutagenesis to the normal DNA as well as the plasmid. Mutagens include: UV light, mutatot strains (e.g. containing error-prone DNA pol etc), EMS or chemical mutagens.
Describe transposons as a method for random mutagenesis including an example
Transposase enzymes exist naturally and they recognise certain sequences in fragments, make a cyclical complex, cut the target DNA and insert the fragment, the gaps are then filled using DNA pol.
MuDel Example
-MuA transposase makes a staggered cut in DNA
-MuDel insert is inserted and the gaps are filled in
-A type II restriction endonuclease cuts the MuDel and a few extra bases out of the target DNA.
1) This gap can be closed resulting in a 3 nucleotide excision which is equivalent to 1 amino acid deletion.
e.g. beta-lactamase found deletions in 2’ structure that survived.
2) A DNA cassette can be ligated into the gap resulting in a domain addition.
e.g. domains were inserted to make beta-lactamase dependent on haem.
3) A set of 3 nucleotides can be inserted resulting in a substitution.
e.g. used in beta-lactamase as part of a scanning mutagenesis procedure.
Describe 2 recombination methods with examples
DNA shuffling - DNAase I fragments 2 or more genetic fragments with around 70% sequence identity. The dsDNA is denatured by heating and then anneals by cooling. This should hopefully get a mixture of the fragments annealing together. DNApolymerase fills in the gaps. e.g. A tree pollen antigen vaccine was produced using 19 genes from 3 different tree pollens, it gave enhanced repsonses.
Recombinant-based PCR - PCR that uses short extension times so that primers may get mixed and different extensions from the parents occur. e.g. stEP was carried out for thermostability on several proteins. The best mutant had a 50-fold increase in half-life at 65 C.
Why use recombination methods to produce a library?
Recombination allows a greater level of diversity than mutagenesis. It can be useful for non-localised and general properties. It produces a broader range so is more likely to find an improvement.
Describe semi-rational methods with an example
Semi-rational methods don’t have that much knowledge but you use what you do know to reduce the size of the library e.g. homolgy models, sequence alignments, structure prediction, structure annotations.
e.g. applying the consensus method to improve penicilin G acylase. This reduced the number of residues suggested to be mutated from 846 to 21.
Describe SCHEMA/RASP with an example
This is a computational method that designs sequences of shuffled variants.
- Collects data from relevant families
- Breaks the sequence into blocks
- Randomly combines assessing for compatibility and the strongest combinations
e. g. This was applied to cellulases with the focus of increasing thermostability. There were 48 genes synthesised and 5 were found to have better half-lives then the parents at 63C.
Describe screening and selecting.
Screening - assaying every single transformant to look for improvements. –> 10^5-10^7 transformants
Selecting - Using a step to remove non-functional of very weak candidates and then assaying the rest. –> 10^9-10^11 transformants
What are the problems with sequence space?
Sequence space is huge, so only a fraction of the library can be sampled, even though you already have to oversample to account for duplicates and underrepresented sequences. High-throughput methods are key to make it effective.
Describe some screening assays
Screening can be fairly with assays that cause a colour change or fluorescence. This can be a direct change from the substrate or from enzyme activity or from a chain of events in the cell. It can be done it multi-well plates or by robots to speed up the process.
Some examples include:
O-nitrophenol - becomes yellow
Methylumbelliferone - fluorescence
Amplex red - reacts with H2O2 and then fluorescence.
Describe flow cytometry
This is a method for screening. Cells are screened for fluorescence using a laser, if the measurement falls into the define range/gate then an electric pulse moves the cell to a different path. The first part is flow cytometry the second is FACS. You can do this on cells or on cells in droplets (good for secreted proteins)
