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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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USES OF ENGINEERED PROTEINS

ADVANDAGES OF PROTEIN ENGINEERING

BIOETHICS

BY

K.PANDI SELVI

II.M.SC.,MARINE BIOTECHNOLOGY



What is protein engineering

What is protein building blocks called engineering?

  • 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.


Cont… building blocks called

  • It is the process of developing useful or valuable proteins.

  • There are two general strategies:

  • Rational protein design

  • Directed evolution


  • Examples

    Examples building blocks called

    • The engineering of fusion proteins has yielded rilonacept.

    • It is used for the treatment of Cryopyrin – associated periodic syndrome.


    • Increased the thermal stability of lysoszyme. building blocks called

    • 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 building blocks called

    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.


    Cont… building blocks called

    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.


    Cont… building blocks called

    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.


    Cont… building blocks called

    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 each compartment of

    • 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.


    Cont… each compartment of

    • The function of a macromolecule is intrinsically linked to atom movements.

    • Molecular dynamics (MD) probes the relationship between molecular structure & function.


    Limitations

    LIMITATIONS each compartment of

    • CPU cost.

    • The availability of a good starting conformation for the macromolecules.

    • MD simulations are not expected to reproduce large conformational rearrangement.


    • Principle:- each compartment of

      • 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.


    • 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.


    • 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 be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    Cont… be applied only to proteins for which a reliable homology model can be obtained.

    • 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 be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    Cont… be applied only to proteins for which a reliable homology model can be obtained.

    • Then transcription of the lactose metabolism genes occurs.

    • Expression, function & silica binding capability of the lac-I derivatives were characterised.


    Diagram describing the function of the lac repressor (LacI). be applied only to proteins for which a reliable homology model can be obtained.


    Characterization of lac i 338 qbp3

    Characterization of lac I-338::QBP3 be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    Fig: Western blot analysis of protein expression be applied only to proteins for which a reliable homology model can be obtained..


    Results conclusion

    RESULTS & CONCLUSION be applied only to proteins for which a reliable homology model can be obtained.

    • 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 be applied only to proteins for which a reliable homology model can be obtained.

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


    • The term be applied only to proteins for which a reliable homology model can be obtained.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


    • In be applied only to proteins for which a reliable homology model can be obtained.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 be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    Cont… be applied only to proteins for which a reliable homology model can be obtained.

    • 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 be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    Cont… be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    CONT… be applied only to proteins for which a reliable homology model can be obtained.

    • They are,

    • To restrict certain types of defensive military research. Ex: toxins.

    • Countries that conduct research involving lethal toxins should pass legislation.


    CONT… be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    CONT… be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    CONT… be applied only to proteins for which a reliable homology model can be obtained.

    • 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.


    • 4. be applied only to proteins for which a reliable homology model can be obtained.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.



    Conclusion

    CONCLUSION established by

    .

    To encourage the beneficial applications of this powerful technology.

    Also, limiting its potential for misuse.

    The appropriate review & oversight mechanisms should be established.


    : established by

    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


    THANK YOU established by


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