Recombinant Engineering in Protein Science

Question 1

Why is recombinant engineering useful?

Answer 1

• can add purification tags
• Prepare plasmid vectors for protein extraction
• express small part of a larger protein complex
• make mutant proteins

Question 2

Why use recombinant proteins?

Answer 2

• Not all proteins are abundant
• Not all organisms can be cultured or are safe (e.g. extremophiles)

Question 3

What are the difficulties associated with recombinant expression in host cells?

Answer 3

• localisation
• toxicity
• folding characteristics
• post-translational modifications

Question 4

What can fusion proteins/tags be used for?

Answer 4

• Purification
• Enhancing expression levels
• Protein detection

Question 5

How can fusion proteins/tags be useful for protein detection?

Answer 5

You can add antibody epitopes, fluorescent proteins, enzymes

Question 6

Why is His-tagging useful?

Answer 6

• Multiple His not found in nature
• High purity in one step
• Interacts with Nickel resin
• Universal technique
• Easy, cheap, reliable

Question 7

Why are synthetic genes useful?

Answer 7

• No need to obtain genomic DNA
• No need for cloning by PCR
• Gene sequence optimized for expression in recombinant host
• Can make artificial proteins

Question 8

How is site directed mutagenesis usually done?

Answer 8

Using mutagenic PCR primers

Question 9

Why is site directed mutagensis useful?

Answer 9

• Powerful way of understanding and engineering proteins
• Rational process which allows for direct testing of amino acid functions

Question 10

What are the problems with site-directed mutagenesis?

Answer 10

• Changing a single amino acid can have dramatic effects
• Helps to have crystal structures
• Results can be tricky to interpret

Question 11

Why should you choose a mutation that deletes part of the side chain or causes isosteric change?

Answer 11

• Any increase in sidechain volume can distort structure
• Small cavities caused by deletions are tolerated

Question 12

Why is it important to avoid creating buried unpaired charges?

Answer 12

Solution energies of ions are very high and unpaired charged groups must be solvated

Question 13

Why is it important to delete the minimum number of interactions?

Answer 13

It is hard enough to interpret the loss of 1 interaction, let alone multiple

Question 14

Why is it important to not introduce any new functional groups?

Answer 14

This can lead to new interactions that can distort protein structure

Question 15

Why should you mutate to alanine if in doubt?

Answer 15

It is a deletion mutation to an inert side chain

Question 16

How was the catalytic activity of subtilisin investigated?

Answer 16

Each of the classical triad of amino acids were mutated to alanine one at a time

Question 17

What happened to subtilisin when any of the active site amino acids were mutated to alanine?

Answer 17

• Had large effect on catalysis
• Binding affinity for substrate slightly increased

Question 18

How was the thermal stability of subtilisn improved?

Answer 18

• Increase number of buried hydrophobic sidechains
• Study thermaphilic homologs and transplant conserved residues
• Increase affinity of binding stabilising Ca²⁺
• Introduce electrostatic interactions