1. Protein engineering involves using genetic manipulations to alter a gene's coding sequence and thus change a protein's properties. This allows improving properties like stability, purity, activity and modifying functions. 2. Stability of enzymes can be increased by adding disulfide bonds or replacing unstable amino acids. For example, mutating xylanase increased its stability at high temperatures. 3. Human pancreatic ribonuclease was modified through site-directed mutagenesis to form a dimer linked by disulfide bonds, creating an effective anti-cancer agent with increased solubility.

1. 1
Lecture- 34

2.  Basic applications - specific mutations in DNA are used
to determine the functions/properties of a DNA sequence
or a protein.
 Commercial applications – Site directed mutagenesis can
be used to get tailor-made proteins for specific
commercial applications.
2

3. 3
Protein Engineering
 Protein engineering involves the use of genetic
manipulations to alter the coding sequence of a
(cloned) gene and thus the properties of the protein
encoded by that gene.
 We can use protein engineering to:
♠ Improve protein stability
♠ Increase protein purity during extraction
♠ Increase protein synthesis
♠ Modify cofactor requirement
♠ Increase enzyme activity
♠ Modify enzyme specificity
♠ Study the function of a protein

4. 4
Improving Protein Stability
 A variety of enzymes are now used in
biotechnology and industry.
 However many enzymes have limited use
because they are denatured on exposure to
conditions which are encountered in
industrial processes e.g. high temperature,
high pH, organic solvents and chemical
solvents.

5. 5
 Protein stability can be increased by creating
a molecule which will not readily unfold
under unfavorable conditions.
 Protein stability can be improved by:
* Adding disulphide bonds
* Replacing labile amino acids
* Reducing the number of free S-H
(sulphydryl) groups.

6. 6
Protein stability can be improved by
changing labile amino acids.
When proteins are exposed to high
temperatures, deamidation occurs.
Deamidation  release of NH3
Asparagine  Asparatic acid
Glutamine  Glutamic acid
The loss of the amide groups may
result in the loss of activity of the
affected enzymes.

7. 7
Increasing Stability of Xylanase
 Current strategies for the production of paper uses
a chemical bleaching step which is essential for
the colour and quality of the paper.
 The bleaching process is used to remove
hemicellulose from the pulp. This bleaching
agent is a potential toxic effluent.
 The bleaching process can be enhanced by using
the enzyme xylanase.
 The use of xylanase reduces the amount of
chemical bleaching agent and the amount of
polluting by-products.

8. 8
 The stage of the process where the enzyme is added
is immediately after hot alkaline treatment.
 In a pulp mill acid is usually added to reduce the
pH to near optima of the enzyme.
 Because of the current trend to reduce the amount
of water during processing the pulp remains hot.
 Therefore a thermostable xylanase is required.
 One attempt to solve this problem was to produce a
modified xylanase (Bacillus circulans) with
increased thermal stability.

9. 9
 Site-directed mutagenesis was used to produce 8
mutants producing xylanase proteins with more
number(s) of S-S bonds to increase stability.
 3 of the mutants were as active as the wild type at
60°C.
 One mutant with an S-S bond between the C and
N terminal ends of the enzyme had twice the
activity of the wild type at 60°C.
 This mutant remained active for 2 hrs while the
wild type lost all its activity after 30 min at 60°C.

10. 10
Making Human Pancreatic Ribonuclease
Antitumorigenic
 Ribonuclease from bull semen (bsRNase) can act as an
antitumorigenic agent.
 This protein is taken up by tumor cells in which it degrades
rRNA blocking protein synthesis.
 The dimeric form of the protein is joined by 2 S-H bridges.
 Antibodies against bsRNase could be produced after its
prolonged use.
 Therefore human pancreatic RNase (hpRNase) was
engineered as an anti-cancer agent

11. 11
 The amino acid sequences of bsRNase and hpRNase are
70% identical.
 The monomeric for hpRNase was modified by site directed
mutagenesis to form a dimer by changing:
╬ Glu 28→ Leu
╬ Arg 31, 33 →Cys
╬ Asp 34 → Lys
 The modified protein was obtained from E. coli by
heterologous expression and was solubilised. This proved
to be a good anti-cancer agent.

12. 12
Human Pancreatic Ribonuclease
Arg
Arg
Gln
Asn
Csy
Cys
Lue
Lys
Lys
Cys
Cys
Lue
A
B
Soluble, unfolded
enzyme
Inclusion body
Enzyme –glutathione
Mixed disuphide
Active dimeric enzyme
1. 6 m guanidine HCl
2. Reduced glutathione
1. Dithioretitol
2. Dialysis
1. Dilute 20 fold
2. Reoxidize

13. 13
Increasing the Thermostability of Triose
Phosphate Isomerase
 Triose phosphate isomerase catalyses the
interconversion of dihydroxyacetone and phosphate to
glyceraldehyde –3 phosphate during glycolysis.
 The enzyme from Saccharomyces cerevisiae consists of
2 identical subunits and each subunit has 2 asparagine
residues which contribute to the thermal sensitivity of
the enzyme.
 Using oligonucleotide directed mutagenesis:
►Asn 14  Ile
►Asn 78  Thr
 The resulting protein had enhanced thermostability as
compared to the wild type protein which was unstable
even at room temperature.

14. 14

15. 15
Study of Intramolecular Bonding in  Interferon
 It was not known which of
the cysteine residues are
involved in intramolecular
bonding.
 A similar molecule 
interferon has 4 Cys residues
at amino acid positions 1 , 29,
98 and 138 with S-S bonds
between Cys 29 and 138,
which is homologous to Cys
31 and 141 of  INF.

16. 16
Increasing the Stability of Subtilisins
 Commonly-used laundry detergents may contain subtilisin
whose wild-type form has a methionine that can be
oxidized by bleach, inactivating the protein in the process.
This methionine may be replaced by alanine, thereby
making the protein active in the presence of bleach.
 The x-ray crystallography structure of the enzyme and the
amino acids involved in the Ca2+ binding were known.
 Oligonucleotide mutagenesis was used to construct a
mutant protein by deleting amino acids 75-83 which are
responsible for Ca2+ binding.
 The next thing to do was to stabilize the modified protein.
 Amino acids of following 4 different regions were changed
: the N terminus (aa 2-5), omega loop (aa 36-44), α
helical region ( aa 63-85) and a β pleated region (aa 202-
222)
 The mutants were assayed for enzyme activity and stability.

17. 17
Increasing the Stability of Streptokinase
 Streptokinase (SK), a 47-kDa protein secreted by
several species of β-hemolytic streptococci, is a
major blood clot-dissolving agent that is used in the
treatment of various circulatory disorders.
 SK combines in equimolar manner with the human
plasminogen (Hpg) molecules forming the complex
SK-HPG which activates HPG to plasmin (Hplm).
Plasmin has the ability to degrade the fibrin matrix of blood
clots but it also cleaves SK at Lys 59 and 386.
 The short in vivo half-life of streptokinase limits its efficacy as
an efficient blood clot-dissolving agent.

18. 18
To make SK less susceptible to cleavage by plasmin,
Lys at 59 and 386 were changed to Glu by site
directed mutagenesis.
Glu was chosen to replace Lys because the length of
the side chain was similar and Glu does not have a +ve
charge.
Both the single mutants and the double mutant
retained their activity.
Furthermore the half lives of all three mutants
increased and the double mutant was 21 fold more
protease resistant .