SITE DIRECTED MUTAGENESIS

& PROTEIN ENGINEERING

 Presented by -

Group – 3

Airen Jahan (1294)

Manotush Vim (1335)
Joyoshrie Karmakar (1347)
Akhi Khanom (1342)
Amina Akter Sathi (1343)
Urmi Khatun (
 What is Protein Engineering ?

A collection of techniques, including but not exclusively gene

mutagenesis, that result in directed alterations being made to
protein molecules, often to improve the properties of enzymes
used in industrial processes.

Protein engineering is merging of several disciplines like

molecular biology, protein chemistry, enzymology, structural
chemistry to alter catalytic or structural stability of protein,
enzyme.
 Objectives of Protein Engineering

 Increasing substrate affinity to enzyme.

 Makes the enzyme thermal tolerant (active at high temp) and PH

stable.

 Enhances the substrate specificity by modifying the substrate

binding site of the enzyme.

 Designing the enzyme to make it resistant to proteolytic

degradation.
 Continued…
 Synthesizing enzyme that is stable and active in non-aqueous
solvents.Changing the enzyme in order to make it independent of
cofactor for its function.

 Improving the stability of the enzyme to heavy metals.

 Fusing the enzymes needed in the reactions to give a final

product.

 Produce hybrid enzymes.

 Make isolation and purification of enzymes simpler.

 Protein Engineering Techniques

Techniques used for protein engineering fall in two basic categories

1. Genetic modifications:
i. Site directed mutagenesis
ii. Localized random mutagenesis

2. Chemical modifications:
i. Chemical mutation
ii. Domain conjugation
 Chemical Modifications
This strategy was necessary because of the absence of cloned genes
or techniques of their manipulation.

1. Chemical Mutation:

 Selective chemical modification of enzymes requires that an amino

acid be uniquely reactive.

 One such residue is the catalytic serine of serine proteases.

 The serine hydroxyl can react specifically with either p-

toluenesulfonyl chloride or phenylmethylsulfonyl fluoride to
introduce a labile leaving group.
 Continued...
 Displacement of the leaving group with varied nucleophiles can
introduce alanine, cysteine, or selenocysteine.

 The loss of serine greatly reduces the hydrolytic activity of these

mutant proteins

 Anhydrotrypsin or anhydrochymotrypsin are useful affinity ligands

for the purification of protease inhibitors.

 The protease variants containing selenocysteine or cysteine

possess relatively unimpaired aminolysis, allowing them to be used
for peptide ligation.
 Continued…
2. Domain Conjugation:

• The conjugation of functionally autonomous units is another strategy

for the synthesis of proteins processing novel function.

• One can also conjugate protein domains directly by genetic fusion at

the amino or carboxy termini, and this will be the focus of a
subsequent section.

• The fusion of domains by chemical cross-linking in vitro, however,

allows the attachment of nonprotein moieties and permits attachment
of domains to any exposed amino acid residue.
 Continued…
• The combination
of protein
subunits during
evolution has
generated
multifunctional
polypeptides that
possess a wide
range of
properties
Figure: Protein engineering by domain conjugation.

Ref: Coombs, Gary S. - Proteins __ Site-Directed Mutagenesis and Protein Engineering (1998, Elsevier)
 What can be Engineered in Proteins ?
 Folding (Structure):

1. Thermodynamic Stability
(Equilibrium between: Native Unfold state

2. Thermal & Environmental Stability

(Temperature, pH, Solvent, Detergents, Salts…)
 Continued…
 Function:

1. Binding
(Interaction of a protein with its surroundings)
How many points are required to bind a molecule with high
affinity

2. Catalysis
(A different form of binding – binding the transition state a
chemical reaction)
Increased binding to the transition state increased
catalytic rates !!!
 Applications of Protein Engineering
Investigative tools: specific mutations in DNA allow the function
and properties of a DNA sequence or a protein to be investigated
in a rational approach.

Xylanase: It is catalytically active at high temperature. Introduction

of disulfide bonds at 1, 2, 3 makes it thermostable and
substantially improves its functional efficiency.

Human β- interferon: It is produced by genetic engineering & exist

as dimer and oligomers which are inactive. This is due to formation
of cysteine. Replacing cysteine with serine reduces free sulfhydryl
groups and makes Human β- interferon more stable.
 Continued…
Insulin: In the neutral solution, therapeutic insulin is present as
zinc containing hexamer. By introducing single amino acid
substitution, insulins were found to be in monomeric state with
good stability and biological activity.

Tissue plasminogen activator: Used to lyse blood clots. Due

to its shorter half-life TPA has to be repetedly administered. By
replacing asparagine residue with glutamine, the half-life can
be increased.