Biotechnology of drugs - Application of
biotechnology in pharmacy
Doc. RNDr. Jan Hošek, Ph.D.
hosekj@pharm.muni.cz
Department of Molecular Pharmacy
FaF MU
Overview of basic biotechnological productions
in pharmacy
➢ Enzymes
➢ Polysaccharides
➢ Steroids
➢ Antibiotics
➢ Antimycotics
➢ Vitamins
➢ Alkaloids
➢ Hormones
➢ Amino acids
➢ Cytokinins
Two approaches
➢ Products obtained by classical
techniques of microbial
biotechnology
➢ Products of recombinant
technologies
Products of classical
biotechnology of
microorganisms
The most common product = ENZYMES
➢ preparation of medicines – antibiotics, steroids, amino acids
➢ medicines – digestive, dissolving blood clots, etc.
➢ diagnostic purposes – part of detection kits
Use of enzymes in pharmacy
Output forms of the enzyme
➢ Enzyme preparation
➢ Enzyme in pure form
➢ Immobilized enzyme
➢ Immobilized cells
Examples of enzymes I.
Proteases
➢ trypsin, chymotrypsin, pepsin, chymosin
➢ papain, ficin, bromelain
➢ bacterial proteases (Bacillus)
➢ proteases produced by fungi (Aspergillus)
Glukosidases
➢ α-amylase, β-amylase
➢ produced by bacteria (Bacillus) and fungi (Aspergillus)
Medicine
➢facilitating the digestion of starch in dyspepsia
➢reduction of meteorism before surgery and in the
postoperative period
Food business
➢production of beer, alcoholic beverages, spirits
➢starch processing into glucose and maltose syrups and
crystalline glucose
➢high fructose syrups
Use of amylases
Origin
➢ pancreas
➢ wheat germ
➢ Aspergillus niger, Rhizopus sp., yeast
Application
➢ part of the digestive system
➢ food industry - cheese production
They catalyze the hydrolysis of triacylglycerols
Lipases
Mechanisms
➢ hydrolysis of penicillin to 6-aminopenicillanic acid
Origin
➢ Escherichia coli, Neurospora crassa, Torula sp.,
Rhodotorulla sp.
Application
➢ production of semi-synthetic penicillins
Hydrolases cleaving bonds other than C-N
Penicilinacylase
Examples of enzymes II.
➢ Cell products capable of inhibiting the
growth of other cells at low
concentrations
➢ The most common producers of G+
bacteria of the genus Streptomyces
➢ Preparation of new ATB by modification
of known ATB, the so-called mutational
synthesis
new ATB
Mutation of microorganism producing ATB + suitable precursor
ANTIBIOTICS
➢ the physiological effect
depends on the exact position
of the substituents in the
basic skeleton
➢ chemical synthesis is very
demanding
Biotransformation of steroids
1) Cultivation of the microorganism
- increase in biomass
2) Addition of steroid, subsequent
biotransformation
3) Isolation into an organic solvent
4) Purification by chromatography
and crystallization
STEROIDES
O
O
11α-hydroxylation
Preparation of 11-a-progesterone
Rhizopus nigricans, R. arrhizus, Aspergillus
ochraceus
11β-hydroxylation
Preparation of cortisol
Curvularia lunata, Cunninghamella blakesleeana
Examples of biotransformations I.
16a-hydroxylation
Preparation of 16a-hydroxy-9a-fluoroprednisolone
(triamcinolone)
Streptomyces roseochromogenes
dehydrogenation between C1-C2
Bacillus lensus, Arthrobacter simplex
Preparation of prednisone, prednisolone,
triamcinolone, 6-methylprednisolone,
dexamethasone…
O
O
Examples of biotransformations II.
Sources
Claviceps purpurea (growing on rye)
Claviceps paspali (submerged cultivation)
Sclerotia of Claviceps
purpurea on rye
Spores on
sclerotium
ERGOT ALKALOIDS
essential animal nutritional factors
Preparation
➢ chemical synthesis
➢ isolation from
natural material
➢ microbial
biosynthesis
➢ biotransformation
VITAMINS
Biotechnologically
produced vitamins
➢ riboflavin (B2)
➢ cobalamin (B12)
➢ ascorbic acid (C)
➢ ergosterole (D2,
D3)
➢ provitamin A
➢ provitamin D
DOI: 10.1533/9780857093547.2.571
➢ Chemical synthesis
➢ Isolation from natural sources
➢ Enzymatic transformations
▪ cultivation of MO containing the
relevant enzyme
▪ cell separation
▪ a substrate intended for enzymatic
conversion is added to the cells
➢ Biosynthetically - cultivation of
microorganism - isolation of
aminoacids from culture
AMINOACIDS
Naturally occurring bacteria
- Corynebacterium
- Brevibacterium
- Micrococcus
Recombinant strains
- Escherichia coli
- Serratia marcescens
Recombinant drugs
➢ the leader is the USA (Food and Drug Administration)
➢ dozens of drugs, all protein-based
➢ hormones
➢ enzymes
➢ hematopoietic growth and coagulation factors
➢ cytokines and interferons
➢ antibodies and their derivatives
➢ vaccines
➢ other products
Types of recombinant drugs
different biological effects
amino acid sequence alteration
changes in pharmacokinetics
broader treatment approaches
Recombinant hormones
y. 1979 - Goeddel et al. - production of
insulin and somatotropin by
Escherichia coli
y. 1982 (28.10.) - insulin - the first
clinically used recombinant hormone
(USA)
(Humulin-R, Eli Lilly and Genentech)
y. 1985 - somatotropin
Recombinant hormones were the first
https://myendoconsult.com/learn/insulin-physiology-and-clinical-applications/
Lispro (HUMALOG)
➢ reversed order of lysine and proline at
positions B28 and B29
➢ production in Escherichia coli
➢ short-acting insulin
Aspart (NOVORAPID)
➢ substitution of proline with aspartic acid
in position B28
➢ Saccharomyces cerevisiae
➢ short-acting insulin
Insulin analogs
Glargin (LANTUS)
➢ addition of 2 arginines to the C-terminus of chain
B and substitution of glycine for asparagine at A21
➢ Escherichia coli
➢ prolonged effect
Detemir
➢ removal of threonine at B30 and
acylation (myristic acid) of lysine at
position B29
➢ prolonged effect
Other types of insulin
https://myendoconsult.com/learn/insulin-physiology-and-clinical-applications/
➢ faster and more regular
absorption from the
subcutaneous tissue
➢ they best mimic prandial
secretion
➢ treatment of patients from 3
years of age
➢ shorter biological effect - lower
risk of hypoglycemia
Advantages over human short-acting insulins
A polypeptide hormone (29 AA)
Effects
➢ glycogenolytic
➢ hyperglycemic
➢ relaxation of GIT smooth muscle
Preparation
➢ Escherichia coli
➢ Saccharomyces cerevisiae
Indication
➢ hypoglycemia
➢ radiological examination – inhibition of GIT movement
Glucagon
A species-specific polypeptide (191 AA)
Effects
➢ growth stimulation
➢ increases proteosynthesis
➢ reduces proteocatabolism
Indication
➢ growth disorders
Preparation
➢ Escherichia coli (since the late 1980s)
Somatotropin
follicle stimulating hormone (FSH)
Two subunits – alfa (92 AA), beta (111 AA)
Production
➢ CHO cells
Indication
➢ anovulatory cycles
➢ amenorrhea
➢ disorders of spermatogenesis
Folitropin
Preparation of medicines
➢ antibiotics, steroides, aminoacids
Drugs
➢ digestive (dissolving blood
clots, treating leukemia)
Diagnostic and R&D purposes
➢ Taq polymerase
➢ Restriction endonucleases
Recombinant enzymes
Epoetin α (165 AA, glycosylated)
Effects
stimulation of blood cell formation
Preparation
mammalian cells
Indication
treatment of anemia
Hematopoietic and growth factors
Interferons α, β, γ
Interleukin IL-2
Effects
immunomodulating effects
Preparation
Escherichia coli
Indication
cancer therapy
Cytokines and interferons
Antibodies
DOI:10.3389/fmicb.2017.00495
There are several production
strategies
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548171/
➢ Methods of reverse genetics
➢ Recombinant subunit vaccines
➢ Production of "virus-like" particles
➢ DNA and RNA vaccines
➢ Vaccines based on viral vectors
Recombinant vaccines
sheep Polly
➢ blood clotting factor IX
➢ treatment of hemophilia
goats
➢ antithrombin III
➢ prevents the formation of blood clots
➢ GTC Biotherapeutics, USA
cows
➢ human lactoferrin
➢ Pharming, Netherlands
Transgenic animals
News of 2024
• Production of
human pro/insulin
in cow milk
Biotechnology Journal, Volume: 19, Issue: 3, First published: 12 March 2024,
DOI: (10.1002/biot.202300307)
Schematic of the lentiviral vector constructed for mammary gland-specific human insulin
expression and restriction map analysis. (A) Schematic of the lentiviral vector constructed. (B)
The restriction map for the vector, L- ladder 1 kb plus (Life Technologies < Waltham, MA); Vconstructed
vector digested with BamHI, ClaI, and XhoI restriction enzymes. The fragment of
the hINS gene was generated by cleavage of the BamHI and XhoI enzymes, while BamHI and
ClaI generated the fragment of the β-casein promoter, and the ClaI and XhoI enzymes
generated the fragment from the original vector pLenti6.2-GW/EmGFP (7.833 kb). The size of the
β-casein promoter used was 5.335 kb and the human proinsulin gene fragment was 1.193 kb.
Transgene analysis. (A) Photo of the transgenic calf. (B) PCR analysis of the
transgene: 1- DNA ladder, 2- transgenic calf, 3- non-transgenic cow, 4- lentiviral
vector constructed. (C) Nonmodified bovine fibroblasts at 5 days incubated with
8 μg mL−1 blasticidin. (D) Fibroblasts from the transgenic calf at 8 days incubated
with 8 μg mL−1 blasticidin.
Isolation and
purification of
recombinant products
Biotechnology products for
therapeutic use must be
precisely specified,
especially when intended
for parenteral
administration.
doi: 10.1002/0471140864.ps0601s80
crystallization, evaporation,
drying, lyophilization
liquid culture treatment of culture fluid
adjustment of pH, heating, addition
of substances causing coagulation
of proteins and cells
cell separation
centrifugation
or filtration
product in fluid
(extracellular)
product in cells
(intracellular)
product isolation
precipitation,
extraction,
chromatography
cell disintegration
Enzymatic
chemical
physical
separation of cell walls
produkt
final product adjustments
centrifugation
or filtration
Isolation and purification of products
Differential centrifugation of E. coli cell lysates. Cells are
broken with a French press or by lysozyme treatment.
Insoluble (inclusion body) proteins, from either the
cytoplasm or periplasm, are located in the low-speed pellet,
which is subjected to preextraction to remove outer
membrane and peptidoglycan material. Inclusion bodies are
extracted from washed pellets with strong protein
denaturants such as guanidine·HCl. The solubilized protein,
which is denatured and reduced (free sulfhydryl residues),
is either directly folded and oxidized (disulfide bonds
formed) or purified before folding. Soluble proteins (from
the periplasm and cytoplasm) are located in the low-speed
and high-speed supernatants. The latter can be used directly
for chromatography, whereas the former requires
clarification by other techniques such as ammonium sulfate
fractionation or membrane filtration.
Curr Protoc Protein Sci. 2015; 80: 6.1.1–6.1.35.
Published online 2015 Apr 1. doi: 10.1002/0471140864.ps0601s80
Localization of secreted and periplasmic proteins in E. coli.
Periplasmic protein produced via a secretion vector can leak
into the medium and be recovered by centrifugation
(supernatant, S1) or filtration. Washing cells with an
isotonic solution such as lightly buffered 0.15 M NaCl or 0.25
M sucrose can also release protein (S2). The
compartmentalized periplasmic proteins are released by
isotonic shock treatment by directly suspending normal cell
paste or plasmolyzed cell paste into hypotonic medium.
Plasmolyzed cell paste is derived by suspending cells in
hypertonic medium and then pelleting. (In hypertonic
medium the cell contracts, separating the inner membrane
from the cell wall, and is said to be osmotically sensitized.)
The hypertonic wash often releases protein (P1). The
supernatant from shocked cells (P2) will contain
constitutive E. coli proteins and the recombinant product.
Osmotically sensitized cells can also be treated with
lysozyme to fragment the outer membrane, thus releasing
periplasmic proteins (P3).The pellet from the lysozyme
treatment contains spheroplasts (cells with fragmented
outer membranes), which are easily disrupted by
detergents, sonication, or hypotonic shock to release
cytoplasmic proteins.
Purification of soluble proteins from bacterial cell and other
cell lysates. Abbreviations for ion-exchange resins are as
follows: CM, carboxymethyl; DEAE, diethylaminoethyl; Q,
quaternary ammonium; S, methyl sulfonate. The order of
preference for the stages of ion-exchange (2) and other
methods (3) is based on the author’s opinion and does not
necessarily represent a consensus view. On the other hand,
the use of a DEAE-based matrix at an early stage (1) is
common practice. Affinity methods (see text and Chapter 9)
can be performed at any stage following clarification of the
lysate.
Folding and purification of inclusion body proteins from E.
coli. The protein is extracted with protein denaturants such
as guanidine·HCl (Gu·HCl), urea, or an organic acid. The
reductant dithiothreitol (DTT) is included to prevent
artificial disulfide bond formation (especially intermolecular
bonds). The denatured protein can be purified by various
methods and then folded, or it can be directly folded.
Typically, some purification (e.g., gel filtration in Gu·HCl)
prior to folding is recommended as it often results in higher
folding yields. Protein folding and oxidation are carried out
concurrently. Disulfide bond formation is catalyzed by lowmolecular-weight
thiol/disulfide pairs such as reduced
(GSH) and oxidized (GSSG) glutathione. GSH/GSSG ratios of
5:1 to 10:1 are normally used, which are similar to those
found in vivo in the endoplasmic reticulum (Hwang et al.,
1992). A cosolvent is included to maintain solubility during
folding. Folded protein is purified if necessary (purification
is usually needed if the protein is directly folded). Gel
filtration is a useful final step for removing aggregated and
or misfolded protein.
Preparation of washed pellets using lysozyme and the
French press. Cells are broken with the French press with or
without prior treatment with lysozyme. After low–speed
centrifugation using a fixed-angle rotor, the contents of the
centrifuge tubes have the characteristics shown. The
contents of tubes A and B are labeled: s, supernatant; lp,
loose pellet; ib, inclusion body protein; and c, unbroken cells
and large cellular debris. The loose pellet material is derived
from the outer cell wall and outer membrane (see text for
further details). After washing the insoluble material (UNIT
6.3), the pellet should consist mainly of the inclusion body
layer (tube C), and the supernatant should be fairly clear.
➢ Quality is usually expressed in terms of
purity and reproducibility.
➢ The purity of pharmaceutical preparations is
usually higher than 99% if they are
administered parenterally.
➢ This also applies to protein therapeutics, for
which the resulting purity is highly
dependent on the purification process.
Contaminating components
➢ Host organism
➢ Product
➢ Process
Origin of contaminating components of
protein pharmaceuticals
➢ Viruses
➢ Host proteins and DNA
➢ Glycosylation variants
➢ Variant N and C termini
➢ Endotoxins from Gram negative bacteria
Contaminating components of the
host organism
➢ Amino acid substitutions and deletions
➢ Protein denaturation
➢ Conformational isomers
➢ Dimers and aggregates
➢ Variants of disulfide bridges
➢ Deaminated proteins
➢ Protein fragments
Contaminating components
originating from the product
➢ Growth medium components
➢ Purification reagents
➢ Metals
➢ Materials of purification columns
Contaminating components created
within the process
Category Method Example
Virus inactivation Heat Pasteurization
Radiation UV irradiation
Dehydration Lyophilization
Crosslinking substances,
denaturation, decomposition
β-propionolacton, formaldehyd,
NaOH, org. solvents, detergents
Neutralisation Specific antibodies
Virus removal Chromatography Ion exchange, immunoaffinity
Filtration nanofiltration
Precipitation cryoprecipitation
How to get rid of virus particles?
Filtration through 15 nm
membranes, which can capture even
the smallest known non-enveloped
viruses such as bovine parvovirus
What is nanofiltration?
➢ Bacteria from cell cultures can be easily
removed by filtration
➢ Work sterile, clean areas (air sterilization)
➢ Work under antibiotics, which are then
difficult to remove !!
Bacteria as a contaminating component
Pyrogens – substances of different sizes with
different structures
➢ Sensitive people - fever with fatal consequences
➢ Remove by ion exchange chromatography
➢ Hot air sterilization of instruments
Mycoplasmas
➢ It changes the characteristics of metabolism, growth,
cell lifespan, etc.
➢ Remove with gentamicin or ciprofloxacin
What else does bacteria do?
➢ When mammalian cells are used, DNA
fragments are present in the medium
➢ What is a safe level?
➢ The European Pharmacopoeia recommends
that the amount of DNA in the final
preparation of the therapeutic protein does
not exceed 100 pg to 10 ng daily dose
depending on the type of culture system
Cellular DNA as a contaminating
component
➢ "Foreign" proteins can be recognized as
antigens → an immune response that the
recombinant protein is not responsible for
➢ Sourced by host cell or medium
➢ Beware of recombinant protein variants!
➢ Detection of contaminating proteins
immunologically
Contaminating proteins
Cell separation
➢ centrifugation
➢ filtration (drum rotary filters)
Cell disintegration
➢ enzymatic: lysozyme
➢ chemical: alkali, detergents
➢ physical: osmotic shock, cell crushing with abrasives, ultrasound
➢ centrifugation
➢ filtration
Separation of cell walls
Isolation of the product from the liquid
➢ Extraction
➢ system of two immiscible solvents
➢ in protein isolation PEG and dextran or PEG and specific salts such as K3PO4 or
NH4SO4
➢ Precipitation
➢ salting out proteins NH4SO4
➢ precipitation with organic solvents (EtOH, acetone, ...)
➢ Chromatographic methods (gel, ionex, bioaffinity, adsorption)
➢ Electromigration methods (electrophoresis, isoelectric focusing,
isotachophoresis)
Final adjustments of the product
Evaporation
Drying
➢ vacuum evaporators
➢ beware of thermolabile substances
➢ for thermolabile enzymes, plate (film)
evaporators are most suitable
➢ removal of water and volatile substances from the product
➢ belt, hazel, drum, spray dryers
➢ fluid-current dryers (blowing the material with warm air) - common in
the pharmaceutical industry
Isoelectric focusing
Two-dimensional electrophoresis
➢ Movement of proteins in a pH gradient after
application of an electric field
➢ Proteins migrate to the so-called "isoelectric point"
➢ Separation by isoelectric focusing - based on
electric charge
➢ Electrophoresis separation - based on size
Chromatography
➢ Separation based on the different permeability of
proteins filling chromatographic columns
Protein separation methods
Mass spectrometry
Antibody detection
➢ Based on the ratio of mass to charge of ionized
molecules (MALDI-TOF)
➢ Polyclonal antibodies
➢ Monoclonal antibodies
➢ Western blot
➢ Immunoprecipitation, immunocytochemistry and
immunohistochemistry
Protein identification methods
➢ It is a collective designation for a group of physicalchemical
separation methods
➢ It is used for the separation and analysis of complex
mixtures of substances
➢ In all types of chromatographic separation, the
molecules of the analyzed substance are divided
between the so-called stationary and mobile phase
➢ The separation is based on the different distribution of
the components of the mixture between the mobile
and stationary phases
Chromatographic methods
According to the purpose of use
➢ analytical chromatography
➢ preparative chromatography
According to the physico-chemical principle
➢ adsorption chromatography
➢ partition chromatography
➢ ion exchange chromatography
➢ gel chromatography
➢ (bio)affinity chromatography
Types of chromatographic methods I.
According to the state of the mobile phase
➢ liquid chromatography
➢ gas chromatography
According to the arrangement of the stationary phase
➢ column chromatography
➢ capillary chromatography
➢ thin layer chromatography (thin layer chromatography)
➢ paper chromatography
Types of chromatographic methods II.
➢ biologically active substances form an extensive
group of compounds with special functions
➢ changes in pH, ionic strength, concentration of
metal ions, cofactors, etc. can result in a large
effect on the isolated biologically active
molecules
➢ in order not to lose their biological activity
during isolation, it is necessary to use the
mildest possible separation methods
Chromatography
➢ low concentration of biologically active substances
➢ a mixture of many similar substances
First stage of isolation = adsorption
➢ biospecific affinity chromatography
➢ at physiological pH values, most proteins are
negatively charged → sorption to annex
Another level of isolation
➢ gel chromatography
➢ electrophoretic methods
Purification strategy – I.
Isolation of a pure biologically active
substance is most often achieved by a
combination of several separation methods
When choosing a purification scheme, care
should be taken not to repeat methods based
on the same separation principle
Purification strategy – II.
It is based on different adsorption of substances on the
surface of the sorbent, forming the stationary phase
➢ Substances that are more strongly bound by sorption forces
under the given conditions are adsorbed in individual
sections more often and for longer than other substances
➢ Stationary phase sorbents differ in polarity or acidity
➢ non-polar - activated carbon, polar acidic silica gel (SiO2)
➢ polar - basic hydrated alumina or magnesium oxide
➢ Mobile phase - solvent mixtures (... chloroform, ethanol, ...)
➢ For gas adsorption chromatography, nitrogen or helium
Adsorption chromatography
It is based on the different solubility of the separated substances in
two different liquids, i.e. on different values ​​of the partition
coefficient (α = cm/cs)
➢ One of the liquids used is the mobile phase, the
other is then anchored on some carrier and thus
forms the stationary phase
➢ Higher value of α = stronger binding to the stationary
component = slower flow through the column
➢ Normal phase = anchored stationary phase is water
➢ Inverted phase = anchored stationary phase consists
of low polar organic liquids
➢ Carriers – SiO2, glass, polymers, starch, cellulose, etc.
Partition chromatography
➢ the basis is the reversible
exchange of ions
between the mobile liquid
and the stationary phase
➢ stationary phase ionexics
(anex or katex)
Ion exchange (ionex) chromatography
Gendeh, Gurmil et al. “Exploration of pH-Gradient Ion-Exchange
Chromatograp y High-Resolution Protein Separations in Biotechnology
d Pr teomics.” (2012).
Initial
stage
Sample
loading
Adsorption
of target
ElutionWash of
colony
product
Course of ion exchange chromatography
https://www.sciencedirect.com/topics/biochemistry-genetics-
and-molecular-biology/ion-exchange-chromatography
Separation of macromolecules based on the
different size of individual substances on a
porous stationary phase (gel filtration)
➢ agarose
➢ cross-linked dextran (Sephadex)
➢ polyacrylamide (BioGel P)
➢ cellulose (Cellufin)
➢ materials based on silica gel or porous glass
Stationary phase - inert porous material saturated with liquid
Gel chromatography
In other words: it is based on the different permeability of the
holes and hollow niches on the particles of the stationary phase
for different sized particles of the partitioned mixture
Gel chromatography
https://www.mblbio.com/bio/g/support/method/chromatography.html
When a mixture of substances passes through
a porous stationary phase, it happens that
➢ small molecules are able to diffuse into the pores of
the matrix and their movement is thus slowed down
➢ large molecules are not captured and pass through
the matrix faster - the larger the molecule, the faster
it passes out of the column
➢ Small molecules are also washed out of the column
by successive washing of the mobile phase
➢ It is important that there are no bonds between the
split solution and the matrix or denaturation of the
split material
Principle of gel chromatography
Small molecules
can "enter" inside
Large molecules cannot
"enter" inside
Cross-linked
gel particles
Gel filtration
https://www.adareng.com/es/articulo/
chromatography-types/n-41
The principle of the isolation method is the interaction of the isolated
protein with a ligand bound to a solid support
based on the exceptional property of biologically active
substances to form strong specific reversible complexes
with other complex-forming compounds, so-called affinity
ligands
enzyme – substrate, cofactor – effector, antibody – antigen,
hormone – receptor, etc.
Ligand =
a compound that forms a biospecific reversible complex
with a given isolated substance
Affinity (bioaffinity) chromatography
Any compound that forms a biospecific reversible complex with
the given isolated substance can be used as a ligand
➢ immobilized pyridine or adenine nucleotides
➢ dyes with an anthraquinone structure
➢ immobilized hemoglobin or casein for proteolytic enzymes
➢ it must contain a functional group that is covalently bound to
a solid support
➢ it must have sufficient affinity for the isolated substance
Ligands in affinity chromatography
http://dx.doi.org/10.1016/j.sbi.2014.04.006
Protein identification
methods
Molecular weight determination method
➢ It divides proteins (peptides) according to the ratio
of their mass and charge
➢ The molecule is first ionized using the MALDI or ESI
(electrospray ionization) method
➢ The resulting ions are drawn into the analyzer by the
electric field, where they are divided according to the
ratio of mass and charge
➢ Computerized data processing follows
Mass spectrometry
matrix-assisted laser desorption ionization time-of-flight
➢ variant of mass spectrometry
➢ peptides are ionized and the mass-to-charge ratio is
determined based on the time-of-flight to the detector
➢ Mw is calculated
and is specific for
each amino acid
Princip of MALDI-TOF
1) There is no need to sequence the
proteins
2) Just knowing the molecular weight
is enough
1) Cannot analyze multimeric proteins
2) The AA sequence should be known
Advantages and disadvantages
➢ Used to study translation results
➢ They arise as a reaction of the organism to an antigen
The region of the antigen recognized by the
antibody is called an epitope
Antibodies as a basic detection and
identification tool
Linear
➢ Antibodies bind to them regardless of
conformation
➢ They recognize, for example, denatured
proteins
Conformational
➢ Antibodies bind to them depending on the way the
polypeptide chain is folded
➢ Antibodies specific for conformational epitopes will
only react with proteins in the native conformation
Epitopes
➢ Identification of polypeptides by antibodies after
separation on denaturing PAGE (alkaline buffer =
proteins acquire a negative charge
➢ Recognition does not take place in the gel, but after
transferring the polypeptides to a membrane
Imunoblotting
Western blotting
Immunoblotting result
➢ It is used to isolate
specific proteins from
protein mixtures using
antibodies
➢ It is used to study
interactions between
proteins
Immunoprecipitation
https://www.leinco.com/immunoprecipitation/