Protein engineering 1

Question 1

What are the three general methods of protein engineering?

Answer 1

Rational design
Data driven
combinatorial

Question 2

What is rational design?

Answer 2

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

Question 3

What is data driven protein engineering?

Answer 3

Engineering proteins based on limited data

- i.e. you know that something doesn’t work

Question 4

What is combinatorial protein engineering?

Answer 4

Randomly mutate and create very large numbers of mutants

Only requires DNA sequence

Question 5

How does Site directed mutagenesis in rational design work?

Answer 5

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)

Question 6

What is scanning mutatgenesis?

Answer 6

Sequentially mutating each amino acid to alanine (or cys/trp) to determine the importance of the amino acids
Arguably a type of rational design

Question 7

Why is Dpnl used over a normal restriction enzyme?

Answer 7

Only targets methylated DNA

Targets 4 base pair sequence not 6, hence forms breaks more frequently and can target more DNA sequences

Question 8

What is site saturation mutagenesis?

Answer 8

You mutate a specific aa site to all possible amino acids

Form of ‘semi-rational’ design

Question 9

What is an example of protein engineering by site saturation mutagenesis?

Answer 9

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

Question 10

What are the two general methods of combinatorial methods of protein engineering?

Answer 10

Random mutagenesis

Recombination of two or more genes

Question 11

What are the two main types of random mutagenesis?

Answer 11

In vivo

PCR based protocols (error prone PCR)

Question 12

What are the two main ways to recombine different genes?

Answer 12

DNA shuffling

PCR-based protocols

Question 13

How is PCR made more error prone?

Answer 13

• 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

Question 14

How is invivo mutagenesis performed?

Answer 14

• 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

Question 15

Are transposons a form of mutagenesis?

Answer 15

yes

Question 16

Briefly explain DNA shuffling?

Answer 16

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

Question 17

Sequences made from DNA shuffling tend to have what percentage homology?

Answer 17

70%

Question 18

In recombination Dependant PCR what is the minimum homology required between the two parental DNA strands?

Answer 18

50%

Question 19

In recombination Dependant PCR what part of the DNA strand do primers bind?

Answer 19

The short extension of the DNA strands

Question 20

How many short extensions does DNA strand in recombination Dependant PCR have?

Answer 20

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

Question 21

Must the number of cross over in recombination Dependant PCR be:

a) even
b) odd

Answer 21

odd

Question 22

How many aa are in Penicillin G Acylase?

Answer 22

766

Question 23

Why was a combinatorial method not used for Penicillin G Acylase stability improvement?

Answer 23

Because the protein is very large

Question 24

What was the Structure Guided consensus Concept used to improve the stability of Penicillin G Acylase?

Answer 24

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

Question 25

What criteria were used to identify possible sites of mutagenesis in Penicillin G Acylase stability improvement?

Answer 25

• 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)

Question 26

How many sites using the criteria for possible sites of mutagenesis in Penicillin G Acylase stability improvement were identified?

Answer 26

21 (out of an initial 109)

Question 27

What causes better thermostability?

Answer 27

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

Question 28

What is SCHEMA/RASPP?

Answer 28

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