seminar sandy
TRANSCRIPT
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PRESENTED BY:
M SANDEEP REDDY.TRINITY COLLEGE OF PHARMACEUTICAL SCIENCES H.NO: 11341P1033M.Pharmacy (Pharmaceutics) 2 nd semester
ENGINEERED PROTEIN BY DNA TECHNOLOGY
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Protein engineering can be defined as the
modification of protein structure with
recombinant DNA technology or chemical
treatment to get a desirable function for better
use in medicine, industry and agriculture.
DEFINITION
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Protein engineering is merging of several
disciplines like:
1.molecular biology.
2. protein chemistry.
3. enzymology .
4.structural chemistry to alter catalytic or
structural stability of protein.
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BASIC ASSUMPTIONS
While doing protein engineering should recognize the following properties of ,
– many amino acid substitution, deletions or additions
lead to no changes in enzyme activity so that they
are silent mutator.
– Protein have limited number of basic structures and
only minor changes are superimposed on them
leading to variation
– Similar patterns of chain folding and domain
structure can arise from different amino acid
sequences with little or no homology
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OBJECTIVES OF PROTIEN ENGINEERING
The objectives of protein engineering is as
follows –
(a) to create a superior enzyme to catalyze the
production of high value specific chemicals.
(b) to produce enzyme in large quantities.
(c) to produce biological compounds(include
synthetic peptide, storage protein, and synthetic
drugs) superior to natural one CONTINUE…
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• Get humanised(chimeric) antibodies with less
immunogenicity .
• Make harmones resistant to attack by antibodies
or stomach enzymes.
• Get more site specific, more potent
biopharmaceutical with altered pharmacological
action.
Over all aim of protien engineering application is to
get funtionally more useful enzymes, antibodies,
harmones, receptor protiens.
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Steps involved in protein engineering
• A study of three dimensional structure of protein
A study of three dimensional structure is the
preliminary steps of protein engineering. And a 3d
structure of protein is produced from the data
generated from X-ray crystallography and NMR
process by protein modeling
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The three dimensional structure of a protein
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•The continuous line represents the primary structure of the
protein.
•You should note that the primary structure has a polarity. That
is, there is a N terminal region of the protein and a C terminal
region.
•The curly sections represent alpha helical regions. These are
components of the secondary structure of a protein.
•The flat sections with arrows represent beta pleated sheat.
These are also components of the secondary structure of a
protein.
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Methods for protein engineering
Chemical modification
methods for protein engineering
Genetic modification
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GENTIC MODIFICATION
• In the gene modification method is to design, develop
and produce proteins with improved operating
characteristics ( increased stability & biological activity)
sometimes creating even novel proteins.
The techniques such as Mutagenesis( site-directed ).&
gene cloning are utilized for this purpose.
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increased stability & biological activity
Performed for thermo stability of proteins, their industrial application and therapeutic use can more appropriately met.
It includes:
• ADDITION OFSULIFIDE BONDS
• CHANGE OF ASPARAGINE TO OTHER AMINO
ACIDS
• REDUCING THE FREE SULFHYDRYL GROUPS
• SINGLE AMINO ACID CHANGES.
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ADDITION OF SULIFIDE BONDS
Significantly increase in the thermostability of enzymes.
However , the addition al disulfide bonds should not interfere
with the normal enzyme function.
The new protein with added disulfide bonds does not readily
unfold at high temperatures.
Examples: T4-Lysozyme, Xylanase.
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CHANGE OF ASPARAGINE TO OTHER
AMINO ACIDS
If asparagine and glutamine present in protein when heated,
ammonia is released amino acids convert to aspartic acid and
glutamic acid. Protein may refold and loss of biological
activity.
Example: triosephosphate isomerase.
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REDUCING THE FREE SULFHYDRYL GROUPS
The protein or enzyme stability and its activity can be
increased by reducing the number of sulfhydryl groups.
Convert Cys to another amino acid (serine?)
• reduce dimerization.
• maintain activity of enzyme.
Examples: Human β- interferons
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SINGLE AMINO ACID CHANGES.
• Some of the recombinant proteins can be improved in
their stability and biology activity by a secondgeneration
variants. These have been frequently achieved by a
single amino acid changes.
Examples: α1 Antitrypsin, insulin, tissue plasminogen
activator, hirudin.
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The other process of gene modification are-
(a) In vitro mutagenesis using synthetic
oligonucleotides.
(b) Synthesis of complete modified gene de novo
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Synthetic oligonucleotides is used for invitro
mutagenesis. In this method a small oligonucleotides
primer containing the desired modification is first
synthesized. It is then hybridized to the appropriate site
and cloned gene and then the rest is replicated using
DNA polymerase enzyme, so that the rest remains
unaltered. This approach is actually used to modify the
active site of the tyrosyl-tRNA synthetase
(a) In vitro mutagenesis using synthetic oligonucleotides.
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Complete gene in some cases have been chemically
synthesized in the form of several oligomers (e.g. genes for
insulin, somatostain and interferon), that are ligated in correct
order to produce a complete gene. The sequence of the
synthetic gene can be designed in a modular fashion to get the
desired function.
(a) Synthesis of complete modified gene de novo
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De novo design of proteins: The attempt to choose an amino acid sequence that is unrelated to any natural sequence, but will fold into a desired 3-D structure with desired properties.
ComputerModeling Gene
construction
Protein productioncharacterization
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Chemical modification of enzymes
The protein synthesized under the control of gene sequence in
a cell undergo post-transitional modification. This leads to
stability, structural integrity, altered solubility and viscosity of
individual proteins.
for e.g. Enzyme-PEG conjugates. An enzyme L-
asparaginase has antitumour properties but is toxic with a
life time of less then 18hrs thus reducing its utility.
Continue……….
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L-asparginase can be modified by polyethene glycol
derivatives to produce PEG-asparginase conjugates, which
differ from the native enzyme in the following way (i) it
retains only 52% of the catalytic activity of the native. (ii) it
become resistant to proteolytic degradation. (iii) it doesn’t
cause allergy.
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Achievements of protein engineering
A number of proteins are known now where efforts have been
made to know the effects of site specific mutagenesis
involving substitution of one or more amino acids.
Insulin- it consist of A and B chains are linked
by C-peptide of 35 amino acids. It was shown that a sequence
of 6 amino acids for c-peptide was adequate for the linking
function. Continue….
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cytochrome c – A phenylalanine residue has been
identified to be non-essential for electron transfer but is
involved in determining the reduction potential of the
protein.
Trypsin- It could be redesigned to have altered
substrate specificity.
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Acetylcholine receptor. This protein is involved in transport, of
acetylcholine through. the membrane. Specific regions of this
protein involved in acetylcholine binding and channel formation
have been, identified.
Human beta interferon.
Removal of one of the three cysteine residues' I led to an
improvement in stability of the enzyme.
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Substantial progress has been made in the of engineered
protein by DNA technology . Observations from Genetic
modification and Chemical modification , Regarded as
redesigning nature rather than copying nature. Protein
engineering and genetic engineering are the complementary
fields to each other the successes in protein engineering will
transform the way we make foods, drugs and chemicals..
Design of protein engineering using DNA technology
remains challenging: Though some solutions have been
found, few guarantees of success currently exist.
Conclusion
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1. Kuhlman, Brian; Dantas, Gautam; Ireton, Gregory C.; Varani, Gabriele; Stoddard, Barry L. & Baker, David (2003), "Design of a Novel Globular Protein Fold with Atomic-Level Accuracy", Science 302 (5649): 1364–1368,
2. Looger, Loren L.; Dwyer, Mary A.; Smith, James J. & Hellinga, Homme W. (2003), "Computational design of receptor and sensor proteins with novel functions", Nature 423 (6936): 185–190.
References
3. www.molecular-plant-biotechnology.info
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THANK U