Protein engineering

Protein Engineering by Directed Evolution

Ben Mair

Enzymes are biological catalysts

Vital area of biotechnology

Can be used to create more sustainable and energy efficient pathways to reactions

Existing biological proteins are being engineered to meet commercial needs

Protein Function

Protein Structure

Polypeptide sequence ‘folds up’, mainly due to hydrogen bonding and dipole interactions

Prosthetic groups

Wikimedia, http://commons.wikimedia.org/wiki/File:Haemoglobin-3D-ribbons.png, (Accessed February 2015).

DNA Structure

Gene Splicing

Rational Engineering Irrational Engineering

View proteins as a sum of modular components

View proteins as a whole

Involves changing genetic sequence at specific points to give rise to desired outcome in protein

Involves random recombination of multiple homologous protein genes, followed by screening for any improved mutant proteins

Beneficial modification requiresgreat understanding of bio-molecular interactions

Leaves beneficial changes up to chance

Rational Vs. Irrational Protein Design

DNA Replication

Random recombination of genes leads to good diversity

However large population of mutant proteins no longer functional

Compromise with rational design is made by mapping important intramolecular interactions in protein

Directed Evolution

SCHEMA Energy of Disruption

𝐸𝛼𝛽 =

𝑖∈𝛼

𝑗∈𝛽

𝑐𝑖𝑗𝑃𝑖𝑗

Where c accounts for broken interactions and P accounts for probability of disruption

Matrix algorithm used (RASPP)

SCHEMA-RASPP technique has enabled productive results from 55% parental homology

C. A. Voigt, C. Martinez, Z. Wang, S. L. Mayo and F. H. Arnold, Nature Struc. Bio., 2002, 9, 553-558.

Cytochrome P450 from Bacillus megaterium modified to convert aliphatic alkanes into secondary alcohols

Turnover rate increased (× 50) over 5 generations of homologous recombination and screening

Active site more complementary to favourable substrates, allowing increased productivity

Exemplar Research: Cytochrome P450

Exemplar Research: Cytochrome P450

PNAS, http://www.pnas.org/content/99/10/6725/F8.expansion.html, (Accessed February 2015).

Not an economical way to produce alcohols:

Translation of gene to protein has to be done in vivo. Protein’s modifications make it toxic to cell (production must be regulated)

NADPH required as electron source for reduction of P450, which is very expensive

Exemplar Research: Cytochrome P450

Limited knowledge on existing proteins

Difficulty scaling up enzyme reactions for commercial potential

Restricted access to protein and gene libraries between institutions

Current Problems with Directed Evolution