Protein Engineering - definition, application, scope methods uses as per PCI syllabus.

1. Protein engineering
By S.D.Mankar
Assistant Professor
Department Of Pharmaceutics
Pravara Rural College Of Pharmacy,Pravaranagar
S.D.Mankar
1

2. One of the most exciting aspects of genetic engineering is to
design, develop, and produce proteins with improved
operating characteristics ( increased stability, improved
kinetics etc)
Besides sometimes creating even novel proteins.
The techniques such as site- directed mutagesis and gene
cloning are utilized for this purpose.

3. Increasing the stability and biological activity of proteins
By increasing the half lives or thermosatbility of enzyme/
protein their industrial applications or therapeutic uses can
be more appropriately met.
Some of the approaches for producing proteins with
enhanced stability are described below.
Addition of disulphide bonds:
Significant increase in the thermostability of enzymes is
observed by adding disulphide bonds should not interfere
with the normal enzyme function.

4. In general, the new protein with added difulphide bonds
does not readily unfold at high temp., and further it is
resistant to denaturation at non-physiological conditions (
high pH, presence of organic solvents).
These characteristics are particularly important for industrial
applications of certain enzymes.
E.g. T4 lysozyme:-
This an enzyme of bacteriophages t4.
Good success has been achieved in introducing disulphide
bonds in T4 lysozyme.

5. This was done by changing 2, 4, 6 amino acids to cysteine
residues to respectively form 1, 2, or 3 disulphide bonds.
T4 lysozyme with 3 disulphide bonds is very stable with good
biological activity.
E.g.-Xylanase:-
 used in industry for mfg of paper from wood pulp.
Catalytically active at high temp.
Introduction of disulphide bonds make it thermostable ans
substantially improves its functional efficiency.

6. Changing asparagine to other amino acids:-
At high temp, the amino acids asparagine and also
glutamine are likely to undergo deamidation(releasing
ammonia) to respectively form aspartic acid and glutamic
acid.
These alterations are often associated with changes in the
protein folding and loss of biological activity.
E.g.triosephosphate isomerase:-
Dimeric enzy mes with identical subunits, each one having
asparagine residues which are termosensitive.

7. It is used to introduce threonine or isoleucine in place of
asparagine at position 14 & 78.
New enzyme found to be thermostable.
Reducing the free sulfhydryl groups:-
Sometimes, the presence of free sulfhydryl groups in more
numbers may be responsible for the low activity of the
protein.
In such case, the protein or enzyme stability and its activity
can be increased by reducuing the number of sulfhydryl
groups.

8. Human beta interferon:-
The antiviral activity of human beta interferon produced by
E.Coli by genetic engineering was found to be only 10 % of
the original glycosylated form.
Further IFN-B was found to exist as dimers & oligomers
which are almost inactive.
This problem was successfully overcome by replacing
cysteine residues by serine.
So it reduces free sulfhydryl groups.
By above process IFN-B with increased stability & good
biological activity can be produced.

9. Single amino acid changes:-
Some of the recombinant protiens can be improved in their
stability and biological activity by a second generation
variants. These have been frequently achieved by a single
amino acid change.
E.g. it inhibit action of neutrophile elastase( it damage lung
tissue leads to emphysema).
By binding with elastase α1 antitrypsin prevent its action
In this process cleavage between serine and methionine is
formed whcich make α1 Antitrypsin poor inhibitor.

10. Problem overcome by replace methionine at 358 positionby
valine.
New enzyme used in α1 Antitrypsin deficiency.
Insulin
In neutral solution is mostly present as a zinc-containing
hexamer
By inroducing single amino acid substitutions,insulin found
to be monomeric state with good stability and biological
activity.

11. Tissue plasminogen activator(tPA):
a. Tissue plasminogen activator is therapeutically used to
lyse the blood clots that cause myocardial infraction.
b. Due to its shorter half- life (around 5 min) ,tPA has to be
repeatedly administered.
c. By replacing asparagine residue (at position 120) with
glutamine ,the half -life of tPA can be substantially
d. This is due to the fact that glutamine is less glycosylated
then asparagine and this makes difference in the half-life of
tPA.

12. Hirudin:
a. Hirudin is protein secreated by leech salivary gland ,and is
strong thrombine inhibitor it acts as an anticoagulant
b. By replacing asparagine with lysine,the potency of hirudin
be increased several folds.

 Dihydrofolatereductase:
a. The enzyme dihydrofolatereductase (DHFR) catalyse the
conversion of 7,8-dihdrofolate to 5,6,7,8-tetrahydrofolate .
b. The latter coenzyme is closely involoved in one carbon
metabolism,which ultimately results in the synthesis of nucleic
acids and amino acids.

13. c. The inhibition of DHFR will restrict the growth of tumor cells.
d. By employing size directed mutagenesis,by replacement of
glysine by alanine was found to produce DHFR which is
completely inactive.

 T4 lysozyme:
a. Replacement of glycine by any other amino acid the protein
structure, in general decreases the stability.
b. On the other hand proline residues increase protein stability .
c. The T4 lysozyme substitution of glycine(at position 77) by
alanine and alanine (at position 82)by proline are found to
increase the enzyme stability.

14. Improving kinetic properties of enzymes:
a. It is possible to improve the functional activities of
enzyme by improving the kinetic properties through
oligonucleotides directed mutagenesis.
b. This is particularly required for enzymes with individual
and therapeutic applications.
Subtilisin:
a. Subtilisin is major industrial enzyme secreted by gram
positive bacteria .
b. Is is a serine protease very widely used as an enzyme
detergent.

15. The reason being that methionine line closed to active sides
get oxidized,making the enzyme inactive.
e. Logically replacement of methionine by others amino
acids will solved the problem
 Modifying metal requirement of subtilisin :
I. The enzyme subtilisin binds to calcium and get stabilized
III. they bind to calcium and the enzyme becomes inactive.
IV. This problems can be solved by oligonucleiotides directed
mutagenesis .

16. Asparaginase:
a. This is an enzyme used in control of leukemia.
b. Intravenous administration of asparaginase cleaves
asaparagine to aspartate .
c. Thus,the asparaginase with a low Km value has to be
seleted for thearapeutic use in control of leukemia.
Tyrosilt-RNAsynthetase:
The enzyme tyrosil t-RNA synthetase has been modified
from Bacillus stearothermophilus with substrate binding i.e.
Km value. This enzyme catalyses two reactions to give
tyrosine t-RNA.

17. Tyrosin + ATP Tyrosyladenylate +PPi
Tyrosyladenylate +t-RNA Tyrosine t-RNA + AMP
Restriction endonuclease
There are 2500 restriction endonucleases are known. Most of the
enzyme recognize same sequence on DNA for action, only about
200 have different recognition site.
Types of restriction endonucleases
Frequent cutters- recognizes sequence of 4-6bp.
 Rare cutters- recognizes sequence above4 8 bp.
Using protein engineering technique, restriction endonucleases
have been suitably modified to produce rare cutters.

18. Protein engineering by use of gene families
The recent development in protein engineering is DNA
shuffling(molecular breeding).
 It can be applied to protein if belong to known protein
family.
 Technique involve isolation of gene from each species, then
creation of hybrids in different combnation.
 26 species were mixed to create a library while applying
approach to subtilisin.
04 were found improved properties.

19. Protein engineering through chemical modification
Chemical modification of proteins have increased the
stability of proteins to high temp. and organic solvents.
 Mostly used cross linker is glutaraldehyde, which stabilizes
protein in solutions.
Certain proteins like insulin, hemoglobin ,
phosphofructokinase have been stabilized using
glutaraldehyde.

20. Protein engineering- ever expanding field
Techniques of protein engineering are rapidly expanding
offering newer developments of protein/ enzymes for
industrial and therapeutic purpose.
The challenge of biotechnologist specialized in protein
engineering is ‘how do I achieve the modification I wish to
make.’