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USES OF ENGINEERED PROTEINS ADVANDAGES OF PROTEIN ENGINEERING BIOETHICS

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USES OF ENGINEERED PROTEINS ADVANDAGES OF PROTEIN ENGINEERING BIOETHICS. BY K.PANDI SELVI II.M.SC.,MARINE BIOTECHNOLOGY. Proteins are polymeric molecules constructed from twenty building blocks called aminoacids. Proteins have diverse functions.

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slide1

USES OF ENGINEERED PROTEINS

ADVANDAGES OF PROTEIN ENGINEERING

BIOETHICS

BY

K.PANDI SELVI

II.M.SC.,MARINE BIOTECHNOLOGY

slide2

Proteins are polymeric molecules constructed from twenty building blocks called aminoacids.

  • Proteins have diverse functions.
  • It can be transport molecules receptors for specific molecules on cell surfaces, enzymes, antibodies & structural proteins.
what is protein engineering

What is proteinengineering?

  • Chemical or genetic alteration of a protein in order to alter its function.
  • Alteration of gene can involve changing specific base-pairs or the introduction of a new piece of DNA into the existing DNA molecule.
slide4

Cont…

    • It is the process of developing useful or valuable proteins.
    • There are two general strategies:
  • Rational protein design
  • Directed evolution
examples

Examples

  • The engineering of fusion proteins has yielded rilonacept.
  • It is used for the treatment of Cryopyrin – associated periodic syndrome.
slide6

Increased the thermal stability of lysoszyme.

  • Improved binding of small molecules to protein receptors on cell surfaces.
  • Altered specificity of DNA – binding proteins & specific metabolic enzymes.
  • Improved biological properties of enzymes.
protein engineering

Protein Engineering

Recombinant DNA techniques in E.coli:-

The expression of heterologous genes in bacteria is the simplest means for obtaining large amounts of desired proteins.

Ex: E.coli

Due to availability of promoters there is efficient production of recombinant proteins.

slide8

Cont…

E.colican readily to grown to high cell density.

This helps in the production of therapeutic & industrial proteins on a large scale.

E.coli, gram negative bacteria contains two membrane.

slide9

Cont…

It is divided into three compartments:

Cytoplasmic

Periplasmic

Extracellular space

The mode of gene expression affects the location of protein produced.

High cell density culture techniques for E.colihave been developed to improved productivity.

slide10

Cont…

Many proteins expressed in E.coli were documented intracellularly & biological activity must be recovered.

Advances in protein refolding, translocation & roles of molecular chaperons & foldases have made possible the design of recombinant E.coli strains.

They strengthen the dominant status of E.coli as a host for production of recombinant protein.

advantages disadvantages

Advantages & Disadvantages

  • Study of the dynamical behavior of macromolecules
  • Principle:-
    • Everything that living things do can be understood in terms of the Jiggling & Wiggling of atoms, Richard Feynman,1963.
slide13

Cont…

  • The function of a macromolecule is intrinsically linked to atom movements.
  • Molecular dynamics (MD) probes the relationship between molecular structure & function.
limitations

LIMITATIONS

  • CPU cost.
  • The availability of a good starting conformation for the macromolecules.
  • MD simulations are not expected to reproduce large conformational rearrangement.
slide15

Principle:-

    • In silico structure based ligand design is becoming a very alternative to high throughput invitro methods.
    • It can be broken into two main stages:
    • To estimate the binding mode of the small ligand on the protein surface.
    • Ligand modifications that can be expected to increase its affinity for the targeted macromolecules.
slide16

Three steps are used in structure – based & fragment – based approach:

  • Selected molecular fragments are connected to form putative ligand.
  • The ligand candidates are examined for synthetic feasibility & their free energies of binding are estimated.
  • Positions & orientations for a series of molecular fragments are determined in the known structure of the macromolecular target.
slide17

In silico structure – based drug design approaches should be applied only to proteins for which a reliable homology model can be obtained.

  • The approach can not able to provide a good ligand in a single shot, independently of any experimental contribution.
  • Optimization are necessary to converge to a good ligand.
use of engineered proteins for organization of nanostructure

Use of Engineered proteins for Organization of Nanostructure

  • The E.coli lac repressor (Lac I) regulates the production of proteins involved in lactose metabolism.
  • Short peptide sequences can be inserted without affecting the normal function of protein.
  • The sequence for the inorganic silica binding motif, QBP3 was inserted into a permissive site at residue 338 of the E.coli lac repressor.
slide19

Cont…

  • After PCR screening for the QBP3 insert, six candidates were sequenced.
  • Western blot analysis to these constructs showed good protein expression.
  • Beta – galactosidase assays indicated Lac I clones maintained normal function.
  • Engineered proteins that bind both DNA & inorganic compounds can be utilized to arrange nanostructure.
introduction

INTRODUCTION

  • DNA binding protein lac repressor is used for regulation.
  • Lac I normally function as a tetramer.
  • In the presence of lactose, allolactose binds to the tetramer inducing a conformational change.
slide21

Cont…

  • Then transcription of the lactose metabolism genes occurs.
  • Expression, function & silica binding capability of the lac-I derivatives were characterised.
characterization of lac i 338 qbp3

Characterization of lac I-338::QBP3

  • Protein expression of the clone was determined by western blotting.
  • Beta – galactosidase assays were carried out to determine DNA binding activity.
  • Silica binding assays were done.
  • The ability of protein to bind to silica was measured.
  • Protein was detected using the lac I antibody on a western blot.
results conclusion

RESULTS & CONCLUSION

  • Two Lac I -338::QBP3 clones were isolated & characterised.
  • Smaller Lac I proteins were more susceptible to degradation.
  • Beta – galactosidase assays showed good repression activity.
bio ethics

BIO ETHICS

It is a philosophical study of ethical controversies brought about by advances in biology & medicine, life sciences, biotechnology, politics, law & philosophy.

slide27

The term Bioethics was coined in 1927 by Fritz Jahr,who “anticipated many of the argument & discussions now current in biological research involving animals”.

  • Bios mean life
  • Ethos mean behavior
slide28

In 1970, the American biochemist Van Rensselaer Potter also used the term "global ethics,"

  • It represents a link between biology, ecology, medicine and human values in order to attain the survival of both human beings and other animal species
principles

Principles

  • The first areas addressed by modern bioethicists was that of human experimentation.
  • In 1974, The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research was introduced basic ethical principle.
  • The conduct of biomedical and behavioral research involving human subjects.
slide30

Cont…

  • In 1979, the fundamental principles announced in the Belmont Report namely, autonomy, beneficence and justice have influenced the thinking of bioethicists across a wide range of issues.
bioethics in protein engineering

Bioethics in protein engineering

  • Advanced protein-engineering techniques require a high level of expertise training & experience.
  • Technologies such as bioinformatics, solid-phase protein synthesis & industrial microbiology are diffusing worldwide.
  • It makes the development & production of engineered toxins increase.
slide32

Cont…

  • Fusion toxins might also be interest for warfare or terrorism.
  • Protein toxins, like botulinum & ricin have applications in therapeutics & biomedical research.
  • To prevent the misuse of protein toxins several policy are undertaken.
slide33

CONT…

  • They are,
  • To restrict certain types of defensive military research. Ex: toxins.
  • Countries that conduct research involving lethal toxins should pass legislation.
slide34

CONT…

  • It should require all academic institutions & private companies that work with such materials to be registered & licensed.
  • Scientific personnel who are planning to work with toxins should undergo a security.
slide35

CONT…

  • 3.The scientific community should promote professional codes of conduct & other forms of self regulation.
    • Scientists should educate themselves & the next generation about their legal & ethical obligations.
slide36

CONT…

  • All researchers, should be encouraged to alert the appropriate authorities if they become aware of suspicious activities.
  • Whistleblowers from any nation or institution should be protected from retribution.
slide37

4.Every country that is engaged in the engineering of protein toxins should establish a national biosecurity board.

    • This board should block the funding of specific projects or to constrain the publication of sensitive scientific results.
    • Regulations governing the registration & licensing of facilities should be harmonized internationally.
slide38

This might be undertaken by a technical working group established by WHO.

  • In areas of research on human & animal pathogens pose biosafety & biosecurity risks warranting regulation, the engineering of protein toxins raises similar concerns.
conclusion

CONCLUSION

.

To encourage the beneficial applications of this powerful technology.

Also, limiting its potential for misuse.

The appropriate review & oversight mechanisms should be established.

slide40

:

BOOK SOURCE:

Sandhya mitra.,Genetic engineering principles &

Practice., First edition (1996)., Macmillon limited, Delhi.

Helen kreuzer.,Adrianne massey., Recombinant DNA &

Technology.,ASM press.

NET SOURCE:

en.wikipedia.org/wiki/protein_engineering

en.wikipedia.org/wiki/bioethics

mbel.kaist.ac.kr/lab/research/protein_en.html

www.unil.ch/pmf/page 48309.html

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