To give knowledge about topic.

1. BRIEF INTRODUCTION TO PROTEIN
ENGINEERING
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Prepared by:
Ms. Harshada R. Bafna.
M. Pharm (Quality Assurances)

2. Introduction
 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 or in pharmaceuticals and other
application.
 Protein engineering is the process of developing useful or industrial important
proteins.
 Protein engineering involves synthesis of new proteins or to make changes in the
existing protein sequence or structure to achieve desired function.
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3. Fig 4.1: Basic principle of protein engineering
Definition:
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.
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4.  To create a superior enzyme.
 To develop more stable and catalytic efficient enzymes.
 To produce biological compounds (include synthetic peptide, storage protein, and
synthetic drugs) superior to natural one.
 Get humanised antibodies with less immunogenicity.
 Get more site specific, more potent biopharmaceutical with altered pharmacological
action.
 Over all aim of protein engineering application is to get functionally more useful
enzymes, antibodies, hormones, receptor proteins.
 Change the substrate binding sites to increase specificity.
 Change the thermal tolerance and pH stability.
objectives
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6. 1. Rational design approach
Techniques of proteinengineering
 It involve ‘site-directed mutagenesis’ of protein.
 Rational design play main role in protein engineering and manipulate
different processes such as regenerative medicines, protein delivery system,
tissue engineering etc.
 It improved by modification in the site directed mutagenesis technique,
protein-protein interaction and protein 2D, 3D structure modeling.
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8. 2. Site-directed mutagenesis
 Site-directed mutagenesis is ‘in-vitro’ technique which allow introduction of
specific amino acid into a target gene.
 It involve the change of cloned target DNA either by deletion, substitution or
insertion of same host cell.
 This host cells are used for production of functional protein.
 The single-prime method is simplest method of site directed mutagenesis.
 It involve ‘in-vitro’ DNA synthesis with chemically synthesized
oligonucleotide (7 to 20 nucleotide) that carries a base mismatch with the
complementary sequence.
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9.  A single stranded clones of the wild type gene is produced by using M13 phage
based vectors.
 The hybrid clone as a primer in the presence of DNA polymerase, synthesizes
second DNA strand.
 The slightly mismatched duplex recombinant plasmid is used to transform
bacteria.
 The duplex DNA replicates in bacterial cell and produce either wild type or
mutant plasmid (Show in fig.4.3).
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11. 3. Chemical modification
 Chemical modification is most widely used and had more important before
advances in site directed mutagenesis.
 In this method, functional group on side chain of natural enzymes may be
changed or part of original protein modified and replaced.
 Protein modification is used for increase the stability of enzyme to high temp.
and organic solvents.
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12. Increasing thestabilityand biological activityof proteins
The industrial application or therapeutic uses of enzymes/proteins can be
appropriately brought into use by increasing their half-lives or thermostability.
Proteins with enhanced stability can be obtained by following methods:
1. Addition of disulfide bonds
2. Changing asparagine to other amino groups
3. Single amino acid changes
4. Improving kinetic properties of enzymes
5. Reducing the free sulfhydryl group
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13. 1. Additionof disulfide bond
 Introduction of disulfide bonds significantly increases the thermostability of
enzymes.
 The disulfide bonds added should not disturb the normal functioning of enzymes.
 The new protein obtained after the addition of disulfide bond does not unfold at high
temperatures and also does not denatures at non physiological conditions (i.e high pH
and presence of organic solvents).
 Eg: T4 lysozyme, xylanase
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14. 2. Changing Asparagineto other amino acids
 The amino acids Asparagine and glutamine undergo deamination (i.e., release
ammonia) to form aspartic and glutamic acid, respectively at high temperature. These
alterations are associated with changes in protein folding and loss biological activity.
 Triose Phosphate isomerase- This is a dimeric enzyme with identical subunits, each
having two thermosennsitive asparagine reduces which undergo deamination.
3. Reducing the freesulfhydryl group
 The presence of a large number of free sulfahydryl groups (contributed by cysteine
residue) may lower the activity of proteins
 In case the stability and activity of the protein or enzyme can be improved by reducing
the number of sulfhydryl groups.
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15. 4. Single aminoacid changes
 The stability and biological activity of recombinant proteins can be improved by a
second generation variant. This is achieved by a single amino acid change.
5. Improvingkinetic propertiesof enzymes
The functional activities of enzyme can be improving their kinetic properties through
oligonucleotide directed mutagenesis.
 This is required for enzymes having industrial and therapeutically benefits.
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16. Application of proteinengineering
 Protein engineering is used for cancer treatment studies. Pre-targeted radioimmune
therapy is potential cancer treatment as pre targeting minimises radiation toxicity by
spreading the rapidly cleared radionuclide and the long circulating antibody.
 protein engineering techniques are also used for producing therapeutic proteins.
Therapeutic proteins are used to treat patients suffering from cancers, heart attacks,
strokes, cystic fibrosis, diabetes, anemia and heamophilia.
 Protease, amylase and lipase are important enzymes for industrial food and detergent
applications.
 Protease are commonly used for food industry in milk clotting, flavours and low
allergic infant formulae.
 Lipase are used for stability and chees and flavours application in food industry
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17.  Amylases are commonly used for scarification of starch and bread softness in food
industry.
 Protein engineering are used for improvement of microbial strains and their
enzymes in bio-remediation applications.
 Redox protein and enzyme can be modified by protein engineering to be used as
Nano devises for biosensing.
 The activity or properties of food-processing enzymes (amylase, lipase) are
improved by protein engineering and rDNA technology.
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