USES OF ENGINEERED PROTEINSADVANDAGES OF PROTEIN ENGINEERINGBIOETHICS

BYK.PANDI SELVIII.M.SC.,MARINE BIOTECHNOLOGY

• 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?• 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…

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

• There are two general strategies:Rational protein designDirected evolution

Examples• The engineering of fusion proteins has

yielded rilonacept.• It is used for the treatment of

Cryopyrin – associated periodic syndrome.

1.Increased the thermal stability of lysoszyme.2.Improved binding of small molecules to protein

receptors on cell surfaces.3.Altered specificity of DNA – binding proteins &

specific metabolic enzymes.4.Improved biological properties of enzymes.

Protein EngineeringRecombinant 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…

• E.coli can 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…

• It is divided into three compartments:CytoplasmicPeriplasmic Extracellular space • The mode of gene expression affects the location

of protein produced.• High cell density culture techniques for E.coli

have been developed to improved productivity.

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 and Disadvantages in the protein production into each compartment of Escherichia coli

Advantages & DisadvantagesStudy of the dynamical behavior of macromoleculesPrinciple:-• Everything that living things do can be understood in

terms of the Jiggling & Wiggling of atoms, Richard Feynman,1963.

Cont…• The function of a macromolecule is intrinsically

linked to atom movements.• Molecular dynamics (MD) probes the relationship

between molecular structure & function.

LIMITATIONS1.CPU cost.2.The availability of a good starting conformation for

the macromolecules.3.MD simulations are not expected to reproduce large

conformational rearrangement.

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:1.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:

1.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

• 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…

• 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• 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…

• 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).

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.

Fig: Western blot analysis of protein expression.

RESULTS & CONCLUSION

1.Two Lac I -338::QBP3 clones were isolated & characterised.

2.Smaller Lac I proteins were more susceptible to degradation.

3.Beta – galactosidase assays showed good repression activity.

BIO ETHICS

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

• 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

• 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

• 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…

• 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

• 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…

• 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…

• They are,1.To restrict certain types of defensive military

research. Ex: toxins.• Countries that conduct research involving

lethal toxins should pass legislation.

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.

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.

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.

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.

• 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.

CONCLUSION1.To encourage the beneficial applications

of this powerful technology.2.Also, limiting its potential for misuse.3.The appropriate review & oversight

mechanisms should be established. .

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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_engineeringen.wikipedia.org/wiki/bioethicsmbel.kaist.ac.kr/lab/research/protein_en.htmlwww.unil.ch/pmf/page 48309.html

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