This seminar discusses protein engineering, which modifies protein structure using recombinant DNA or chemical treatment. The objectives are to create superior enzymes for industrial chemical production and drugs. Proteins must be robust, stable under industrial conditions, and efficiently use non-natural substrates. Protein engineering aims to alter properties like kinetics, thermostability, pH and substrate optimization. It involves studying protein structures, using mutagenesis, selection and recombinant DNA to achieve desired functions.
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
Strain improvement Part II, Generation of mutants producing high level of pri...Renu Jaisinghani
This presentation, describes about various mutants that can be generated by carrying out process of mutation, so that high yielding mutants can be obtained that can be used for industrial production of primary metabolite.
Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”
Mutagenesis; A conventional tool for strain improvement in industry Zohaib HUSSAIN
The strain improvement is the process of improvement and manipulation of microbial strains for the icreasment of metabolic level for industrial applications. The yield of microbial enzymes can be increased by using microbe specific medium for fermentation, improving the fermentation process and strain improvement for higher yield of product.
All these things lead to decrease in cost production. Microbe produce product according to its need therefore there is great need for overproduction. There is tremendous contribution of conventional Mutagenesis for strain improvement. Mutagenesis is important tool for the production of mutants which are capable to produce large product i.e. hyperactive.
Metabolic engineering is a branch of bioengineering where the use of genetic engineering and recombinant technology to modify the metabolism of an organism.
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
A study of effect of Saccharomyces cerevisiae on three substrates namely vanillin, orange peel oil and clove oil. The method uses HPTLC for analysis of biotransformation products.
Strain improvement Part II, Generation of mutants producing high level of pri...Renu Jaisinghani
This presentation, describes about various mutants that can be generated by carrying out process of mutation, so that high yielding mutants can be obtained that can be used for industrial production of primary metabolite.
Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”
Mutagenesis; A conventional tool for strain improvement in industry Zohaib HUSSAIN
The strain improvement is the process of improvement and manipulation of microbial strains for the icreasment of metabolic level for industrial applications. The yield of microbial enzymes can be increased by using microbe specific medium for fermentation, improving the fermentation process and strain improvement for higher yield of product.
All these things lead to decrease in cost production. Microbe produce product according to its need therefore there is great need for overproduction. There is tremendous contribution of conventional Mutagenesis for strain improvement. Mutagenesis is important tool for the production of mutants which are capable to produce large product i.e. hyperactive.
Metabolic engineering is a branch of bioengineering where the use of genetic engineering and recombinant technology to modify the metabolism of an organism.
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
A study of effect of Saccharomyces cerevisiae on three substrates namely vanillin, orange peel oil and clove oil. The method uses HPTLC for analysis of biotransformation products.
Protein engineering is the process of developing useful or valuable proteins.
Protein Engineering is a second generation of recombinant DNA technology.
It involves altering cloned DNA in vitro by novel mutational technique so that translated proteins have slightly altered properties.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
PEPTIDOMIMETICS/SAGAR SHARMA/DEPARTMENT OF PHARMACEUTICAL SCIENCESSagarMudgil1
Peptidomimetics are compounds that mimic natural peptides and proteins in 3D space. They are created by modifying the side chain or backbone of peptides. Peptidomimetics are designed to be highly selective, bioavailable, and metabolically stable.
This comprehensive program covers essential aspects of performance marketing, growth strategies, and tactics, such as search engine optimization (SEO), pay-per-click (PPC) advertising, content marketing, social media marketing, and more
2. PROTEIN
ENGINEERING
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.
3. OBJECTIVES OF PROTEIN
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.
4. RETIONALE OF PROTEIN
ENGINEERING
For industrial application an enzyme,
should possess some characteristics in
addition to those of enzymes in cells.
These characteristics are :-
(1) enzyme should be robust with long
life.
(2) enzyme should be able to use the
substrate supplied in the industry even it
differs from that in the cell.
5. (3) enzyme should be able to work
under conditions, e.g. extreme of pH,
temperature and concentration of the
industry even if they differ from those in
the cell.
6. In view of above, the enzyme should be
engineered to meet the altered needs.
Therefore efforts have been made to
alter the properties of enzymes.
These are some character that one
might have to change in a predictable
manner in protein engineering or
enzyme engineering to get the desired
function :-
7. Kinetic properties of enzyme-turnover
and Michaelis constant, Km.
Thermo stability and the optimum
temperature for the enzyme.
Stability and activity of enzyme in
nonaqueous solvents.
Substrate and reaction specificity.
Cofactor requirements
Optimum PH.
Molecular weight and subunit structure.
8. Therefore for a particular class of
enzymes, variation in nature may occur
for each of the above properties, so that
one may like to combine all the optimum
properties to the most efficient form of
the enzyme.
For an e.g. glucose isomerases, which
convert glucose into other isomers like
fructose and are used to make high
fructose corn syrup vital for soft drink
industries.
9. Basic assumption for protein engineering
While doing protein engineering should
recognize the following properties of
enzymes,
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.
10. 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.
11. The three-dimensional structure of penicillin, for which Dorothy Crowfoot Hodgkin
was awarded the Nobel Prize in Chemistry in 1964. The green, white, red, yellow and
blue spheres represent atoms of carbon, hydrogen, oxygen, sulfur and nitrogen,
respectively.
12. Ribbon diagram of the structure of myoglobin determined with the x-ray
crystallography
13. Pacific Northwest National Laboratory's high magnetic field (800 MHz, 18.8 T) NMR
spectrometer being loaded with a sample.
14. Methods for protein engineering
A variety of methods are used in protein
engineering such as
mutagenesis, selection and recombinant
DNA technology.
15. Mutagenesis
Mutagenesis and selection can be
effectively utilized fro improving a specific
property of an enzyme.
E.g. for E.coli anthranilate synthetase
enzyme is normally sensitive to tryptophan
inhibitor due to feedback inhibition but an
altered MTR2 mutation of E.coli was found
to possess an altered form of enzyme
anthranilate synthetase that is insensitive
to tryptophan inhibition. And thus helping in
the continuous synthesis of tryptophan
without inhibition.
16. Gene Modification
The two process of gene modification are-
(a) In vitro mutagenesis using synthetic
oligonucleotides.
(b) Synthesis of complete modified gene
de novo.
17. (a) In vitro mutagenesis using synthetic
oligonucleotides.
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
18. Synthesis of complete modified gene de
novo.
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.
19. 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. L-
asparginase can be modified by
polyethene glycol derivatives to produce
20. 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.
21. 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.
22. 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.