Protein engineering 1 Flashcards
What are the three general methods of protein engineering?
Rational design
Data driven
combinatorial
What is rational design?
Changing specific aa based on a prior knowledge of structure and function
Generally, works best for changing localised properties – i.e. binding and catalytic activity
What is data driven protein engineering?
Engineering proteins based on limited data
- i.e. you know that something doesn’t work
What is combinatorial protein engineering?
Randomly mutate and create very large numbers of mutants
Only requires DNA sequence
How does Site directed mutagenesis in rational design work?
Using Mutagenic primers to change an amino acid or do a small insertion or deletion
You can then perform PCR using these primers
Use Dpnl to graded methylated DNA strand (i.e. the original parent DNA)
What is scanning mutatgenesis?
Sequentially mutating each amino acid to alanine (or cys/trp) to determine the importance of the amino acids
Arguably a type of rational design
Why is Dpnl used over a normal restriction enzyme?
Only targets methylated DNA
Targets 4 base pair sequence not 6, hence forms breaks more frequently and can target more DNA sequences
What is site saturation mutagenesis?
You mutate a specific aa site to all possible amino acids
Form of ‘semi-rational’ design
What is an example of protein engineering by site saturation mutagenesis?
Βeta galactosidase substrates is galactose. They wanted to make it specific to fructose.
3 sites of ssm
~8000 combinations in total (19^)
mutant found that can catalyse fructose
mutation then made to remove galactose catalysis function
What are the two general methods of combinatorial methods of protein engineering?
Random mutagenesis
Recombination of two or more genes
What are the two main types of random mutagenesis?
In vivo
PCR based protocols (error prone PCR)
What are the two main ways to recombine different genes?
DNA shuffling
PCR-based protocols
How is PCR made more error prone?
- Uses Taq polymerase because it lacks 3’ to 5’ exonuclease proofreading (but only 0.1% error rate)
- Excess Mg2+ or Mn2+
- Unbalanced concentration of dNTPs (ie. More of one than another)
- Higher cycle number – more you copy the more chance for mutations
These additional factors increase mutation rate to 2%
Still a bias to AT to CG mutations
Can use mutazyme II to avoid bias
How is invivo mutagenesis performed?
- Using mutator strains
- UV, EMS, chemical mutagens
You have to provide enough mutagens to overwhelm the cell so that it can’t repair all damage before the next cycle
Are transposons a form of mutagenesis?
yes
Briefly explain DNA shuffling?
Fragment two strands of DNA USING DNASE 1
Apply heat to melt Double stands and hybridise strands Fill in the gaps using Polymerase
Amplify with PCR
Sequences made from DNA shuffling tend to have what percentage homology?
70%
In recombination Dependant PCR what is the minimum homology required between the two parental DNA strands?
50%
In recombination Dependant PCR what part of the DNA strand do primers bind?
The short extension of the DNA strands
How many short extensions does DNA strand in recombination Dependant PCR have?
1
each DNA strand has one and these are on alternate ends in order that only recombined genes have both strands and can be amplified in PCR
Must the number of cross over in recombination Dependant PCR be:
a) even
b) odd
odd
How many aa are in Penicillin G Acylase?
766
Why was a combinatorial method not used for Penicillin G Acylase stability improvement?
Because the protein is very large
What was the Structure Guided consensus Concept used to improve the stability of Penicillin G Acylase?
High homology and high number of sequences in MSA means you have high confidence in conserved regions and so will produce a smaller library and you can have more targeted mutants.
I.e. create MSA and only mutate regions of high conservation that are therefore deemed ‘important’
If low homology and low numbers of sequences you can input PDB structural information to identify further conserved regions
What criteria were used to identify possible sites of mutagenesis in Penicillin G Acylase stability improvement?
- Substitutions only considered if >50% in the consensus
- Substitutions must have a distance of 10Å from active site
- Substitution should not destroy possible stabilizing interactions (i.e., hydrophobic interactions, hydrogen bonds, salt bridges)
How many sites using the criteria for possible sites of mutagenesis in Penicillin G Acylase stability improvement were identified?
21 (out of an initial 109)
What causes better thermostability?
Better core packaging, Increased Salt bridging /Pi-Pi stacking, Increased rigidity (P substitutions), Fewer Thermolabile Residues, “optimised” electrostatics
i.e. there is no one cause for better thermostability
What is SCHEMA/RASPP?
Works out how to break up protein into blocks which wouldn’t effect folding based disrupting the minimum number of side chains interactions.
These blocks can be randomly recombined or based on favourability
Gives information on how to shuffle a protein to get a higher probability of success
