Directed Evolution and Protein Engineering

Question 1

What is directed evolution, explain in the context of protein engineering.

Answer 1

DE consists of forcing biological evolutionary selection steps to optimize the properties of proteins for specific applications.

• Create library of protein variants via random mutagenesis or site-directed mutagenesis
• Protein variants are subjected to selection and screening processes (exposure to antigen, screening via FACS, identifying high affinity variants)
• Variants with desired trait are isolated and become starting point for next round of DE
• Iterative cycles of mutation library-screening-selection to improve the desired protein trait
• Sequencing to identify mutations responsible for desired trait

Question 2

Describe and compare libraries prepared by site-directed mutagenesis and by random mutagenesis.

Answer 2

Site-directed:
- Mutations are introduced at targeted positions and the target region is expected to influence desired protein activity.
- Generated by degenerate oligonucleotides (NNK, NNS) and PCR.

Random:
- Mutations are introduced anywhere on a gene
- Generated by error-prone PCR (altering concentrations of PCR buffers and polymerase) or by recombination
- Generated by recombination (gene fragments recombine via regions of partial homology)

Differences:
- Site: deep diversification at defined regions of gene/protein
- Random: mutations across whole genome, can sample larger sequence space.

Question 3

a) Explain the method of Deep Mutational Scanning (DMS). Include a description of type of mutagenesis library, screening approach, and analysis of data.

b) why did it not predict the Brazilian and African COVID mutations, only UK one?

Answer 3

a) Method using combined DE, Deep seq, and rational mutagenesis
- Large scale data sets generated by systematically mutating subsequent single point positions on aa sequence of protein

1) Mutagenesis library
- Library where each variant has mutated amino acid in different positions
- Yeast display to express mutants on surface

2) Screening
- Testing for affinity, separate binders from non-binders
- See which mutations are beneficial, neutral, or detrimental

3) Sequencing and Analysis
- Deep Seq
- Organize data into sequence-function heatmap
- Each mutation at each position given a functional score

b) DMS studies effects of single point mutations on function, SARS-CoV-2 mutates combinatorially at many positions at a time so DMS missed the escape for E484K.

Question 4

You observe side effects in humans that were not found in mice, what could it be due to? How to avoid it?

Answer 4

The mouse antibody is recognized by the patient’s immune system as a foreign protein. The human body generates human anti-mouse antibody (HAMA) response.

This can be avoided by humanization of the Ab either by:
- Finding regions of human-mouse sequence similarity and replacing the mouse sequence with the human sequence
- Exchanging one region of the Ab at a time, performing DE to improve its affinity/specificity, changing another region etc.