1.
Protein Engineering And Proteome Analysis

2.
Protein Engineering

3.
• Protein engineering is a branch of biotechnology that falls under a
discipline called synthetic biology.
• 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 ,
agriculture , and other fields also.
• Protein engineering is the process of developing useful valuable
protein.
• Protein engineering involves premeditated change of amino acids
and is usually based on the known 3-D structure of a given protein
and its biochemically established catalytic mechanism.
Protein Engineering

4.
History
1- In 1951 Fredrick Sanders discovered
how to determine the sequence of amino
acid .
2- Before this it was thought that protein
have no definite structure. This allowed
for translation to be studied as it provided
the framework for DNA coding of protein.
3- In 1983 Kary Mullis developed the PCR.

5.
Reason To Engineering A Protein
1- To bring changes in catalytic properties .
2- To bring changes in structural properties.
3- Creation of new system .

6.
Objectives / Need of protein engineering
⮚ To create a superior enzyme to catalyze the production of high value specific
chemicals
⮚ To produce enzyme in large quantities.
⮚ To develop useful valuable proteins .
⮚ To get a desirable function for better use in
medicine , industry and agriculture .

7.
⮚ Elimination of allosteric Regulations.
⮚ Improved kinetics properties of
enzymes.
⮚ To produce biological compound
(include synthetic peptides , storage
protein , and synthetic drugs ) superior
to natural one.

8.
⮚ Enhanced substrate and reaction specificity.
⮚ Increased thermostability.
⮚ Alteration in optimum pH.
⮚ Solubility for use in organic solvents.
⮚ Increased / decreased Optimal temperature.
⮚ To speed up the process ( rate of Reaction )
⮚ Increase Protein / Enzymes shelf life.
⮚ To get High quality of product suitability face.

9.
Basic assumption for protein engineering
• While doing protein engineering should recognize the
following properties of enzymes,
o Many amino acid substitution , deletions or addition
lead to no changes in enzymes activity so that they
are silent mutator.
o Protein have limited number of basic structures and
only minor changes are superimposed on them
leading to variation.
o Similar patterns of chain folding and domain
structure can arise from different amino acid
sequences with little or no homology.

10.
METHODS :
A variety of methods are used in protein engineering such as:
• MUTAGENESIS
• PROTEIN ENGINEERING BY USE OF GENE FAMILIES
• PROTEIN ENGINEERING THROUGH CHEMICAL MODIFICATION.
The most classical method in protein engineering is the so-called “rational design” approach which
involves “site-directed mutagenesis” of proteins.
The use of “evolutionary methods” that involve “random mutagenesis and selection” for the desired
protein properties was introduced as an alternative approach.
“In vitro protein evolution systems” are also important methods in protein engineering.
“Flow cytometry”, a powerful method for single cell analysis, is also used in protein
engineering studies.

11.
• Method name Reference(s)
• Rational design (Arnold, 1993)
• Site-directed
mutagenesis (Arnold, 1993), (Antikainen &
Martin, 2005)
• Evolutionary methods/
directed evolution (Arnold, 1993)
• Random mutagenesis (Antikainen & Martin, 2005), (Wong
et al., 2006),
• DNA shuffling (Antikainen & Martin, 2005),
(Jackson et al., 2006)
• Molecular dynamics (Anthonsen et al., 1994)
• Homology modeling (Anthonsen et al., 1994)

12.
The most classical method in protein engineering is the so-called
“rational design” approach which involves “site-directed
mutagenesis” of proteins.

13.
the use of “evolutionary methods” that involve “random
mutagenesis and selection” for the desired protein properties was
introduced as an alternative approach

14.
PROTEIN ENGINEERING BY USE OF GENE
FAMILIES
• This technique involves isolation of gene from
each species and create hybrid for eq-
substillisin ( an enzyme used detergent
industry), genes from 26 species were mixed so
we get 4 types of substilisin enzyme with
improved qualities in different aspect

15.
PROTEIN ENGINEERING THROUGH CHEMICAL
MOdIFICATIONS.
• In this method different chemicals used as
“glutaraldehyde”.
• It acts as protein cross linker.
• It stabilizes the protein in solutions.
• For example, insulin, lactate de hydrogenase

16.
APPLICATIONS:
A variety of protein engineering applications have been reported in the literature.
These applications range from biocatalysis for food and industry to
environmental, medical and nanobiotechnology applications.
Food and detergent industry applications :
Early reports on the importance of protein engineering methods to design new
enzymes for enzyme biotechnological industries date back to 1993 (Wiseman,
1993).
Particularly, the enzymes used in food industry were emphasized as an
important group of enzymes, the industrially important properties of which could
be further improved by protein engineering.
Environmental applications :
Environmental applications of enzyme and protein engineering are also another
important field.
Early reports on enzyme and cell applications in industry and in environmental
monitoring, such as environmental biosensors, date back to 1993.

17.
Medical applications:
Medical applications of protein engineering are also diverse.
The use of protein engineering for cancer treatment studies is a
major area of interest.
Pretargeted radioimmunotherapy has been discussed as a
potential cancer treatment.
By pretargeting, radiation toxicity is minimized by separating the
rapidly cleared radionuclide and the long-circulating antibody.
Advances in protein engineering and recombinant DNA
technology were expected to increase the use of pretargeted
radioimmunotherapy.

18.
PROTEOME ANALYSIS

19.
PROTEOME:
►proteome word coined from 2 words protein & genome by marc Wilkins in 1994.
►proteome refers to the total set of proteins expressed in a given cell at a given time.
►it is defined as the full complement of proteins produced by a particular genome.
►proteome is larger than the genome, especially in eukaryotes, in the sense that there are
more proteins genes.
►the proteome of a cell depends on the cell type,its developmental stage,
environmental/stimuli, nutritional and metabolic status etc.
TYPE OF PROTEOME:
1. CELLULAR PROTEOME
2. COMPLETE PROTEOME
INTRODUCTION

20.
►Proteomics is being applied in both clinical and basic
research.

21.
PROTEOMIC TOOLS TO STUDY
PROTEINS:
☻PROTEIN ISOLATION: 1-Mechanical method
2-Chemical method
☻PROTEIN SEPARATION: Two methods:
1-SDS-PAGE
2-IEF
☻PROTEIN IDENTIFICATION: Use of Mass Spectrometry

22.
WHY PROTEOMICS
• Proteomics is the protein equivalent of genomics and has captured the
imagination of biomolecular scientists worldwide.
• Aimed at determining the identity and quantity of expressed proteins in
cells, their three-dimensional structure.
• For example, it is extensively applied to the study of proteins involved
in carcinogenesis, as well as to discover biomarkers for clinical use,
for screening, diagnosis, staging, prognosis, monitoring response to
treatment, and detection of recurrent diseases.
• The question of how one protein regulates the activity of another by
binding to it requires an integration of structural, functional, and
dynamic information.

23.
From Genomics to Proteomics
• It involves the identification of proteins in the body and
the determination of their roles in physiological and
pathophysiological functions.
• Proteins that are directly involved in both normal and
disease-associated biochemical processes
• Genomics does not predict PTM that most proteins
undergo
Human Genome
Project
Protein compliment of the human
organism.

24.
PROTEOMICS APPLICATIONS
• Most common application of proteomics is protein
identification.
• To accomplish this, we use affinity columns and other
strategies to select for protein targets.
• Characterization of Protein Complexes:
• Proteomics Approach to Protein Phosphorylation
• Proteome Mining

25.
Challenges
• Proteins are more difficult to work with than DNA and
RNA; have secondary and tertiary structure.
• Proteins cannot be amplified like DNA.
• Each proteomics technology can only analyze proteins
within 3–4 orders of magnitude
• Thulasiraman et al. developed the new deep proteome
approach via ligand library beads

26.
Perspectives
• Differential proteomics is a scientific discipline that detects
the proteins associated with a disease.
• Proteomics research permits the discovery of new protein
markers for diagnostic purposes.
• Biomarkers may also be used to help devising an optimal
therapeutic treatment plan for different patient subsets and
to monitor the effect of treatment.
• Proteomics has much promise in novel drug discovery.
• Proteomics makes a key contribution to the development of
functional genomics.

27.
Proteomics Analysis By Mass
Spectrometry

28.
Mass Spectrometry
• Mass spectrometry (MS) is an analytical technique that produces spectra
(singular spectrum) of the masses of the atoms or molecules comprising a
sample of material
• Mass spectrometry is an extremely valuable analytical technique in which the
molecules in test sample are converted into gaseous ions that are
subsequently separated in a mass spectrometer according to their mass-to-
charge ratio (m/z) and detected

29.
Components Of A Mass Spectrometer
❖ Sample input system
❖ Ionization source
❖ Mass analyzer
❖ Detector
❖ Computer based data acquisition and processing system

30.
Mass Spectrum
❖ The mass spectrum is a plot of a relative abundance of the ions at
each m/z ratio.
❖ In most cases, the nascent molecular ions of the analyte produce
fragment ions by cleavage of the bond and the resulting
fragmentation pattern constitute the mass spectrum

31.
Principle And Instrumentation
1. IONIZATION
❖ Ionization is a process of charging a molecule.
❖ The sample molecule must be charged in order to measure them using a
mass spectrometer
❖ The atom is ionized by knocking one or more electrons off to give a positive
ion.
❖ The particle in the sample (Atom or molecules) bombarded with stream of
electron to knock one or more electron out of the sample particles to make
positive ions.

32.
Principle And Instrumentation
2. ACCELERATION
❖ The ions are accelerated so that they all have the same kinetic energy
❖ The positive ions are repelled away from the positive ionization
chamber and pass through 3 slits with voltage in decreasing order.
❖ All the ions are accelerated into freely focused beam.

33.
Principle And Instrumentation
3. DEFLECTION
❖ The ions in the deflected by a
magnetic field according to their
masses.
❖ Different ions are deflected by
the magnetic field by different
amounts.
Amount of deflection depends upon
1. The mass of the ion
2. The charge of the ion

34.
Principle And Instrumentation
4. Detection
❖ The beam of ions passing through
the machine is detected electrically
❖ Ions leaves space in metal by
neutralizing it & the electron in the
wire shuffle along to fill it.
❖ The flow of electrons in the wire is
detected as an electric current
which can be amplified & recorded A continuous dynode particle multiplier detector.

35.
How Mass Spectrometry Works

36.
Applications
• Mass spectrometry is also used to determine the isotopic
composition of elements within a sample. Difference in mass
among isotopes of an element are very small, and less abundant
isotopes of an element are typically very rare,
• Mass spectrometry is an important method for the
characterization and sequencing of the proteins
• Mass spectrometry (MS), with its low sample requirement and
high sensitivity, has been predominantly used in glycobiology for
characterization and elucidation of glycan structures

37.
Disadvantages
• This often fails to distinguish between optical and geometrical isomers and
the positions of substituent in o-, m- and p- position in an aromatic ring
• Also, its scope is limited in identifying hydrocarbons that produce similar
fragmented ions.

38.
Overview of MALDI-ToF
• MALDI-ToF is an instrument used to find molecular mass of a molecular substance through laser
desrption and ionization.
• Analyzes large compounds such as:
• Proteins
• Peptides Oligonucleotides
• Polymers
• MALDI considered a soft analytical technique source

39.
Theory Behind MALDI-ToF
• Sample preparation
• Droplet is dried and form a solid substance
• Droplet is dried and forms a solid substance
• Plate is placed in instrument for analysis
• Sample is found using computer
• Sample analysis
• Laser shot at sample creating plumb
• Energy is transferred to matrix then to sample
• Ions travel through ToF chamber at different speeds
• Ions are detected by detector and sorted according to mass .
• Smaller ion are detected first because they travel faster

40.
The process of MALDI-TOF mass spectrometry

41.
2D Gel Electrophoresis
Two-dimensional gel electrophoresis (2-D electrophoresis) is a powerful
and widely used method for the analysis of complex protein mixtures
extracted from cells, tissues, or other biological samples.
1. This technique separate proteins in two steps, according to two
independent properties: the first-dimension is isoelectric focusing (IEF),
which separates proteins according to their isoelectric points (pI); the
second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
which separates proteins according to their molecular weights (MW).
2. In this way, complex mixtures consisted of thousands of different proteins
can be resolved and the relative amount of each protein can be determined.

42.
Procedure
• The procedure involves placing the sample in gel with a pH gradient, and applying a potential
difference across it. In the electrical field, the protein migrates a long the pH gradient, until it
carries no overall charge. This location of the protein in the gel constitutes the apparent pI of
the protein.
• There are two alternatives methods to create the pH gradient - carrier ampholites and
immobilized pH gradient (IPG) gels. In the Maiman Institute for Proteome Research the IEF
is performed with commercial IPGs for highly reproducible results.
• The IEF is the most critical step of the 2-D electrophoresis process. The proteins must
be solubilize without charged detergents, usually in high concentrated urea solution,
reducing agents and chaotrophs. To obtain high quality data it is essential to achieve low
ionic strength conditions before the IEF it self. Since different types of samples differ in their
ion content, it is necessary to adjust the IEF buffer and the electrical profile to each type of
sample.
• The separation in the second dimension by molecular size is performed in slab SDS- PAGE.
Twelve parallel gels can be separated in a fixed temperature to minimize the separation
variations between individual gels.

43.
RPLC
Reverse Phase Liquid Chromatography (RPLC) is an extremely important subtechnique of HPLC employing
an important subset of the bonded phase chromatography. The technique is easily recognizable since, in
comparison to normal or straight phase techniques, it reverses the polarity of the original adsorbent as
well as the polarity of the mobile phase.

44.
Until the nineteen-sixties, the separation of
'non volatile analytes' was often performed by
paper, and thin-layer chromatography. These
techniques were:
● Slow,
● Lacked sufficient separation power,
● Did not quantitate reliably.
History
new theoretical insights accompanied by important
developments in column packing technology and
chromatographic equipment paved the way for
what is now called High Performance or High
Pressure Liquid Chromatography (HPLC). The new
technique provided:
● Much higher resolution,
● More accurate quantitative results,
● Shorter analysis times in comparison to the
earlier techniques.
Since its introduction HPLC has evolved into an
indispensable tool in many analytical laboratories
and is applied to diverse analytical problems.
Given the advantages and its high separation
potential, RPLC has become (with the exception of
large molecules) the separation mode of choice for
the often simultaneous separation of nonpolar,
polar, and ionic analytes. Not surprisingly,
therefore, RPLC is used in a large and still growing
number of application fields. It has become an
indispensable analytical technique in many areas

45.
Introduction
RPLC is the dominant analytical and preparative technique in many other scientific and
industrial settings as well. The technique is still developing, in spite of its 40+ years of use,
It employs a nonpolar stationary phase (most frequently a hydrocarbon chain chemically
bonded to porous silica particles) and a polar mobile phase constituted by water and at
least a water-miscible organic solvent, which performs as a modifier. RPLC with alkyl-
bonded stationary phases is an extraordinary separation technique that has become the
most popular HPLC mode, representing the vast majority of all HPLC separations
One of the primary factors responsible for the development of RPLC was the need of
separating mixtures containing compounds that were not sufficiently volatile or that lacked
the necessary thermal stability to be analyzed by gas chromatography, such as many polar
and ionic organic and inorganic compounds, drugs, and biomolecules.

46.
Normal phase V/s Reversed phase
Reverse Phase Liquid Chromatography (RPLC) is an extremely important subtechnique of HPLC employing
an important subset of Bonded Phase Chromatography. The technique is easily recognizable since, in
comparison to normal or straight phase techniques, it reverses the polarity of the original adsorbent as
well as the polarity of the mobile phase compared to
Its flexibility and applicability to the separation of nearly all types of analytes have contributed to the
widespread use and popularity of RPLC. The following table summarizes a number of the properties of
straight and reversed phase chromatography in order to highlight the differences.

47.
Advantages of RPLC
● RPLC can separate nearly all molecules with the exception of large molecules.
● A large number of high quality and efficient RPLC stationary phases are available
Combined with the wide spectrum of potential mobile phase mixtures, this offers a
range of selectivity adequate to solve nearly any separation problem.
● Water frequently forms an inexpensive, non-toxic and major part of the mobile phase.
In addition, many samples to be analyzed are soluble in aqueous-organic mixtures.
● once a suitable column has been selected, the absolute retention and selectivity
(retention relative to other solutes) can be easily manipulated using several
experimental factors, such as the percentage of the organic modifier and the
concentration of a wide range of additives (such as ion pairing reagents, surfactants,
ionic liquids, and chiral selectors), pH, and temperature.
● the easy implementation of gradient elution
● the compatibility with aqueous samples
● the possibility of separating nonpolar, polar, and ionic compounds in the same run;

48.
Stationary phase
Nonpolar modified stationary phases are by far the most
important column packings in RPLC. In the majority of such
phases, alkanes or other non polar functional ligands are
covalently bonded to a support material or substrate. The
length of the carbon chains bonded to a substrate is typically
somewhere between C1 and C22. Among these stationary
phases, octyl (C-8) and octadecyl (C-18) modified silicas are
well-known and are often used as standard column packings.
Columns and stationary phases for RPLC must provide:
1. Retention and selectivity sufficient to solve a specific
analytical problem.
2. High efficiency in order to achieve acceptable
resolution
3. Satisfactory column longevity resulting from adequate
chemical, mechanical and thermal stability.
4. Excellent batch to batch and column to column
reproducibility and long term availability.
5. Acceptable pressure drop
Mobile Phase
Many RPLC protocols use a blend of water and a
miscible organic solvent (e.g., acetonitrile or
methanol) as the mobile phase. The purpose of
the organic solvent is to maintain the polarity at a
low enough level for the solute to dissolve in the
mobile phase and yet high enough to facilitate
the binding of the preferred molecule with the
stationary matrix. In some scenarios, ion-pairing
agents such as trifluoroacetic acid may also be
added. Once the molecule of interest is bound to
the column matrix, it is made to dissociate from it
by decreasing the polarity further by increasing
the concentration of the organic solvent in the
mobile phase.
This process of varying the amount of organic
solvent in the mobile phase to separate a
molecule of interest is called a gradient elution.

49.
Substrate types
RPLC stationary phases can be manufactured from different susbtrate types, for example:
inorganic oxides such as silica, alumina, zirconia; or organic polymers like styrene-divinylbenzene,
methacrylates and graphitized carbons. In general, substrates for RPLC phases should meet the
following major requirements:
1. High mechanical strength adequate to withstand pressures of 500 bar and higher.
2. Uniformly shaped pores with a narrow size distribution (Ängstrom range) and sufficient
porosity,
3. Large and homogeneous surface area
4. Uniformly shaped particles (μm range) with a narrow particle size distribution.
5. No swelling or shrinking upon exposure to solvents.
6. Chemical and thermal stability under different experimental conditions.
7. High purity; free of metals contamination.
Silica meets nearly all of these requirements and, in fact, is a dream substrate for the synthesis of
RPLC phases. Silica falls short only in terms of chemical stability since it begins to dissolve at a pH
of approximately 7.5 . New synthesis and modification techniques, however, have resulted in
greatly improved silica substrates and stationary phases thereof showing high chemical and
thermal stability.

50.
Silica can be produced with many different morphologies, porosities
and surface areas. For example, silica can be manufactured:
● In well defined irregularly or spherically shaped particles of
specific sizes and of controlled porosities and pore diameters.
● In addition to these more common morphologies, silica is also
widely used for the manufacture of monolithic, non- porous and
pellicular RPLC stationary phases.
The particle size of the beads is based on the specific requirements of
the separation. Larger bead size implies larger capacities and
potentially lesser pressures. Large-scale preparative processes benefit
by using beads of diameter greater than 10 μm; small-scale preparative
and analytical separations, on the other hand, benefit with beads sizes
in the 3–5 μm range.
The choice of ligand is often governed by the principle that the greater
the hydrophobicity of the molecule to be purified, the less is the need
for the ligand to be hydrophobic. This principle has also led to the rule
that chemically synthesized peptides and oligonucleotides are best
separated with C18 ligands and that protein and recombinant peptides
are better separated with C8 ligands.

51.
Reference
• Ahuja, SK., Ferreira, GM. & Moreira, AR. (2004). Utilization of enzymes for
environmental applications. Critical Reviews in Biotechnology, Vol.24, No.2-3,
pp.125-154, ISSN: 0738-8551
• Akoh, CC., Chang, SW., Lee, GC. & Shaw, JF. (2008). Biocatalysis for the
production of industrial products and functional foods from rice and other
agricultural produce.
• Journal of Agricultural and Food Chemistry, Vol.56, No.22, (November 2008),
pp.10445-10451, ISSN: 0021-8561