Protein Engineering

Dr. Rajesh Patel

 What is protein engineering?
Directed mutagenesis of genes to improve
protein performance

WHY?
selection of naturally occurring enzymes
requires compromises
 How do we change it?
• Detailed knowledge of protein structures
– Crystallography
– Sequencing
• Computer modelling of protein reactions
• Site directed mutagenesis
– Insertion of specific sequences
• Random mutagenesis
– Error prone PCR
• Construction of synthetic genes and hence
synthetic proteins
 What can we change?
• Kinetic parameters
• Stability
– Thermal
– pH
– Non-aqueous solvents
• Specificity
• Optimal conditions
– Thermal
– pH
• Cofactor requirements
• Protease resistance
• Allosteric regulation
• Molecular weight and subunit structure
 Changing Kinetic Parameters
• Change active site
• Change other portions of protein
• Requires detailed knowledge of reaction
steps
– Binding, orientation, activation,
monosubstrate into the active site, reaction
(number of steps), release
Reaction Sequence for Lipase
Lipase
 Changing Specificity
• Changing substrate specificity
• Changing chain length specificity
• Enantiomeric specificity
• Positional specificity
 Changing stability
• Unfolding two stage process
– Reversible local unfolding followed by global
irreversible unfolding and aggregation

• Controlled by reaction kinetics of system

and hence by G
 Factors affecting Stability
• Van der Waals’ interactions
• Hydrogen bonds
• Salt bridges
• Torsion potentials
• Bond stretching
• Planarity of conjugated systems
• Pi–pi stacking
• The entropy of water (this is by far the biggest term of all, and
probably the least well understood)
• Interactions with ions
• Loop tension
• Helix dipole interactions
• Disulfide bridges
 Techniques
• Systems are poorly understood
• Approximations made
– Computer models of statistically determined
force fields
– Modeling of thermodynamic cycles to cancel
out unknowns
• Software available e.g. WHATIF that will
substitute amino acids at different points
and look at local effects
Thermolysin-like proteases
Strategies for protein design
and engineering
Strategies for protein design and
engineering
• Mutagenesis methodology
Rational design
Directed evolution
Advanced design approach
• Screening or selection
• A guide to protein evolution
Directed evolution: random mutagenesis + high-
throughput screening
Advantages
- Do not require any information of structure and mechanism.
- Mutation in random position can be achieved easily.
- Emergence of novel HTS process for increasing sequence
diversity will extend the application.
drawbacks
- need an efficient selection or screening system for large library
- move within narrow fitness landscape, suitable for fine-tuning
of protein properties, not for new function.