PRESENTED BY:

M SANDEEP REDDY.TRINITY COLLEGE OF PHARMACEUTICAL SCIENCES H.NO: 11341P1033M.Pharmacy (Pharmaceutics) 2 nd semester

ENGINEERED PROTEIN BY DNA TECHNOLOGY

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

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.

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

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…

• 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.

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

The three dimensional structure of a protein

•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.

Methods for protein engineering

Chemical modification

methods for protein engineering

Genetic modification

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.

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.

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.

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.

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

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.

The other process of gene modification are-

(a) In vitro mutagenesis using synthetic

oligonucleotides.

(b) Synthesis of complete modified gene de novo

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.

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

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

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……….

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.

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….

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.

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.

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

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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