Affinity chromatography with eg, of protien purification AND tissue plasminogen activator

1. AFFINITY CHROMATOGRAPHY
AND
TISSUE PLASMINOGEN ACTIVATOR
Presented by :- Pramod kumar
M.sc Microbial biotechnology
MDU Rohtak

2. INTRODUCTION
• Affinity chromatography is a method of separating biochemical
mixtures based on a highly specific interaction such as that
between antigen and antibody, enzyme and substrate or
receptor and ligand.
• These interactions which are typically reversible are used for
purification by placing one of the interacting molecules referred
to as affinity ligand onto a solid matrix to create a stationary
phase while a target molecule is in the mobile phase.

3. Principle
• The stationary phase consists of a support medium (e.g. cellulose beads) on
which the substrate (or sometimes a coenzyme) has been bound covalently, in
such a way that the reactive groups that are essential for enzyme binding are
exposed.
• As the mixture of proteins is passed through the chromatography column,
those proteins that have a binding site for the immobilised substrate will bind
to the stationary phase, while all otter proteins will be eluted in the void
volume of the column.

4. Elution
• Once the other proteins have all been eluted, the bound
enzyme(s) can be eluted in various ways:
• by increasing the ionic strength of the buffer, e.g. with a sodium
chloride gradient, so weakening interactions between the
enzyme and the immobilised substrate
• by changing the pH of the buffer, again weakening interactions
between the enzyme and the immobilised substrate
• by adding a high concentration of substrate (or a substrate
analogue) to the elution buffer, so that there is competition
between the free and immobilised substrate for the enzyme
protein

6. •REQUIREMENTS

7. CHROMATOGRAPHIC MEDIA
• A matrix in its use here is a substance,usually in bead form to which a
specific ligand is covalently bound.
• In order to for the matrix to be effective it must have certain characters:
• 1)It must be insoluble in solvents and buffers employed in the process
• 2)It must be chemically and mechanically stable..
• 3)It must be easily coupled to a ligand or spacer arm onto which the
ligand can be attached.
• 4)It must exhibit good flow properties and have a relatively large surface
area for attachment

8. IMMOBILIZED LIGAND
• The ligand can be selected only after the nature of the macromolecule to
be isolated is known.
• When a hormone receptor protein is to be purified by affinity
chromatography, the hormone itself is an ideal candidate for the ligand.
• For antibody isolation ,an antigen or hapten may be used as ligand.
• If an enzyme is to be purified,a substrate analog,inhibitor,cofactor,or
effector may be used as a the immobilized ligand.

9. ATTACHMENT OF LIGAND TO MATRIX
• Several procedures have been developed for the covalent
attachment of the ligand to the stationary phase.all procedures for
gel modification proceed in two separate chemical steps:
• 1)Activation of the functional groups on the matrix and
• 2)Joining of the ligand to the functional group on the matrix.
• A wide variety of activated gels is now commercially available.the
most widely used are described in the following:

10. • CYANOGEN BROMIDE-ACTIVATED AGAROSE
• This gel is especially versatile because all ligands containing primary amino groups
are easily attached to the agarose.since the gel is extremely reactive,very gentle
conditions may be used to couple the ligand.
• 6-AMINOHEXANOIC ACID(CH)-AGAROSE AND 1,6-DIAMINOHEXANE(AH)-AGAROSE
• These activated gels overcome the steric interference problems by positioning a six
carbon spacer arm between the ligand and the matrix.
• Ligands with free primary amino groups can be covalently attatched to CH-
agarose,whereas ligands with free carboxyl groups can be coupled to AH-agarose.
• CARBONYLDIMIDAZOLE(CDI)-ACTIVATED SUPPORTS
• Reaction with CDI produces gels that contain uncharged N-alkylcarbamate groups.
• EPOXY-ACTIVATED AGAROSE
• This gel provides for the attachment of ligands containing hydroxyl,thiol,or amino
groups.

11. • SELECTION OF A GEL OR LIGAND
• Many type of matrix-ligand systems are
commercially available and cost are reasonable
so time can be saved by purchasing preactivated
gel for direct attachment of ligand.
•

12. • BUFFER
• Buffer is used for formation of complex between a
matrix and ligand.as slight change in ionic
concentration weakens the interactions between
them.
• AFFINITY ELLUTION
• In this method a selective substance added to the
buffer causes selective elution of bound
macromolecule-ligand complex.resulting in elution of
desired macromolecule.
•
• CHAOTROPIC AGENTS
• If gentle and selective elution methods do not release
the bound macromolecule then mild denaturing
agents can be added to the buffer.the most powerful
agents are urea,guanidine

13. APPLICATIONS
• 1)It is used for isolation and purification of all biological
macromolecule.
•
• 2)It is used to purify nucleic acid,antibodies,enzymes.etc
• 3)To notice which biological compounds bind to a
particular substance.
• 4)to reduce a amount of substance in a mixture

14. Histidine Tagged antibody purification
• A widely employed method utilizes immobilized metal-affinity
chromatography (IMAC) to purify recombinant proteins containing a
short affinity tag consisting of poly-histidine residues.
• IMAC is based on the interactions between a transition metal ion
(Co2+, Ni2+, Cu2+, Zn2+) immobilized on a matrix and specific amino
acid side chains.
• Histidine is the amino acid that exhibits the strongest interaction
with immobilized metal ion matrices, as electron donor groups on
the histidine imidazole ring readily form coordination bonds with the
immobilized transition metal.

16. • Following washing of the matrix material, peptides containing
polyhistidine sequences can be easily eluted by either
adjusting the pH of the column buffer or adding free
imidazole to the column buffer.
• In general, a six histidine tag is an appropriate choice for the
first trial when adding a novel polyhistidine tag to a protein.
• Polyhistidine affinity tags are commonly placed on either the
N or the C terminus of recombinant proteins.

21. Matrix
• Commercially available matrices like nickel-nitrilotriacetic
acid (Ni2+
NTA)17 and Co2+ carboxylmethylaspartate (Co2+-
CMA) are used.
• The Ni2+
NTA matrix has a binding capacity of 5–10 mg
protein/ml of matrix resin and a high binding affinity for the
six residue polyhistidine tag at pH 8.0.
• The Co2+–CMA matrix has a somewhat lower affinity for the
polyhistidine affinity tag than the Ni2+–NTA resin, resulting in
elution of the tagged proteins under milder conditions.
• The Co2+–CMA also has been reported to exhibit less
nonspecific protein binding than the Ni2+–NTA resin,
resulting in higher elution product purity.
• The binding capacity of the Co2+–CMA resin is also about 5–
10 mg of protein/ml of resin

23. Advantages and disadvantages
Advantages Disadvantages
Simplification Tag removal
Increase solubility Interfere with structure
Prevent proteolysis
Detection

25. Tissue plasminogen activator
• Tissue plasminogen activator (abbreviated tPA or PLAT) is
a protein involved in the breakdown of blood clots.
• It is a serine protease (EC 3.4.21.68) found on endothelial cells, the
cells that line the blood vessels.
• As an enzyme, it catalyzes the conversion
of plasminogen to plasmin, the major enzyme responsible for clot
breakdown.
• Because it works on the clotting system, tPA (such as alteplase,
reteplase, and tenecteplase) is used in clinical medicine to
treat embolic or thrombotic stroke.
• Use is contraindicated in hemorrhagic stroke and head trauma.

26. Tissue Plasminogen activatorTissue Plasminogen activator

27. Overview of action of tpa

28. 1.1 Production of Tissue plasminogen activator1.1 Production of Tissue plasminogen activator
• Therapeutic potential of tPA as a fibirinolytic agent drove the
development of its large scale production.
• The host cells used in production of tPA are decribed
below.........
i. Mammalian cells
Further two types:-
a) Non recombinant producers.
b) Recombinant producers.
ii. Bacteria
iii. Yeast and fungi.

29. 12/10/15
a) Non recombinant producers.a) Non recombinant producers.

30. b) Recombinant producersb) Recombinant producers
• Mammalian cells inserted with copies of human tPA gene
have generally been better producers then other cell line.
• Suitable engineered mammalian cells will produce human tPA
. Cloning and expression in mouse fibroblast,
rat myeloma, and CHO cells as in recombinant human
mylanoma cells containig additional copies of tPA gene.
• CHO cells are generally superior producers then most of cells
studed

31. 12/10/15
b)Recombinant producersb)Recombinant producers

32. 2. Bacteria2. Bacteria
• Tha bacterium E.coli has been used for tPA production from
tPA gene from Bowes melanoma.
• The expretion in bacteria eliminated the risk of expressing
tumor associated proteins and the risk of animal viruses.
• tPA from bacterial origin is a non-gycosilated single chain
polypeptide within the cell as insoluble denatrured inclution
bodies consitituting 5-10%of total protein.

33. 2.Yeast and Fungi2.Yeast and Fungi
• Production of tPA in Saccharomyces cereviase has been
proven possible but the yield has been low .
• Production of tPA from Aspergillus nidulans has been
reported.
• yield has been obtained upto 1 mg / ltr .
12/10/15

34. 
Large scale cultivation of host cells.
Large scale cultivation of host cells.
• The science of growing cell and microbes to produce large
quantities of pharmaceuticals and chemical compound underwell
specified condition is called fermention.
• The genetically engineered host cells for production of recombinant
protiens must be cultured in large quantities ( up to kilograms ).
• Fermentation is applied on cells grows in suspension .
• These include most prokaryotes – E.coli.
• Lower Eukaryotes – Yeast.
• Somtimes mammalian cells aprroved by FDA- CHO.

35. Description Batch size (litres) Time
Laboratory shake flask 0.1 1-2
Large flasks 1-2 2-4
Batch fermenter 50 4-6
Batch fermenter 2500 6-8
Batch fermenter 25000-100000 10-16
 Fermentation and alternative production techniques are
carried out in four different ways.
1. Batch process.
2. Fed batch process.
3. Chemostate process
4. Perfusion configration.
The batch size cell culture and estimated time required for fermentationThe batch size cell culture and estimated time required for fermentation

36. 1.2 Downstream processing of Tissue pa1.2 Downstream processing of Tissue pa
1. Regardless of the type and configuration of the cell and bioreactor
combination used to prepare recombinant product, cells are harvested
at their optimal growth and viability to ensure the highest yield per unit
of cell culture.
2. The downstream processing starting with the biomass cells and medium
harvested from the fermentation and mass cultivation process. Can be
devided in to 4 stages.
(i) Solid liquid sepration or clarification
(ii) Concentration .
(iii) Purification and
(iv) Quality control & assurance analyses.
Recombinant product must meet purity and sterlity standerds and must
below acceptable cellular or microbial contamination( i.e. Less then 0.5
endotoxin )

37. i. Solid-liquid sepration or clarification.i. Solid-liquid sepration or clarification.
1. Sepration of cells from culture media or broth is the primary step in
collecting the product from cells (solid) or medium (liquid).
2. If the protein is interacellular in form of inclusion bodies then the cells
are isolates and then distrupted to collect the recombnant protein
fraction.
Some techniques are used for the sepration of cells.
A. Centrifugation.
B. Filteration
C. Floculation

38. 12/10/15
i.Solid-liquid sepration or clarification.i.Solid-liquid sepration or clarification.
2. Animal cell culture.
a. Sepration of cells from broth is done using microfilteration . With addition
of aprotinin to supress the process of two chain molecule.
b. Cross flow Ultra filteration is used to concentrate the broth. Zinc chelate
chromatography can also done.
3. Microbial culture...
a. From fungus e.g. Aspergillus nidulans.
• There is the product is secreted out in the broth and recovery from
culture broth is similar to the animal celll culture.
b. From yeast and bacteria e.g. Saccharomyces cerevisiae and E.coli .
• The product is intra cellular so cell disrupsion and debris removal are
required prior to purification.

39. (ii) Concentration of recombinant protein(ii) Concentration of recombinant protein
• Concentration is required for the proceeding of next step
i.e. Purification using chromatographic conditions.
• in this step we have to reduce the volume and thereby
increase the recombinant protein concentration.
Some strategies are followed......
(i) Heat assisted evaporation stratgies
(ii) Precipitation.
(iii) Membrane sepration using ultrafiltraton technologies.

40. (iii) Purification(iii) Purification
The purification process of a protein includes the following
steps.
1. Intermidiate purification
• In this stage, increased purity is achived through removal of most
contaminating proteins, nucleic acid, endotoxins and viruses.
• This is acomplished by chromatography.
• In this technique proteins binds to solid matrix support with various
functional group to provide hydrophobic, ion exchange, and affinity
interactions.
• The purified product after processing is composed of more then 90%
recombinant proteins.

41. Purification of recombinant tpa
• The first purification matrix was zinc chelate- Sepharose CL4B.
• The column was equilibrated with 0.02 M phosphate, 0.15 M
sodium chloride, 0.01% Tween 80, pH 7.4 (PBS/TW) and
medium was passed through. Then washed with :-
• (i) PBS/TW,
• (ii) 0.02 M Tris, 1 .O M sodium chloride, 0.01% Tween 80, pH
7.4,
• (iii) 0.02 M phosphate, 0.3 M sodium chloride, 0.01% Tween
80, pH 7.4, and was finally eluted with
• (iv) 0.02 M phosphate, 0.3 M sodium chloride, 0.05 M
imidazole, 0.01% Tween 80, pH 7.4.

42. • The eluate of the imidazole containing buffer was
passed directly onto a column
• After the equivalent of two bed volumes of the
column had passed through the lysine Sepharose
CL4B column the flow from the former was stopped.
• column was then washed sequentially with
(i)PBS/TW,
• (ii) 0.02 M Tris 0.5 M sodium chloride, 0.01% Tween
80, pH 7.4, and
• (iii) 0.02 M Tris 0.5 M sodium chloride, 0.5 M L-
arginine, 0.01% Tween 80, pH 7.4.
• All stages of the chromatography were carried out
at 5-10°C.

43. Concentration
• After eight successive purifications individual
pools of rt-PA were thawed and mixed and
were concentrated using stirred-cell
ultrafiltration. The ultrafiltered retentate was
stored at - 40°C.

45. (iii) Purification(iii) Purification
When the product is produced as inclusion bodies in bacteria E.Coli.
• Then we have to addition step have to be taken for the renaturation of
protein in a series of low yield steps.
i. Inclution bodies are washed with a solution of 5 M urea and a surfactant
such as triton(2%) followed by the dissolution in a denaturantes such as
guanidine hcl (7 M) .
ii. The dissolution buffer may contain a reducing agennt such as ;beta-
marcaptoehanol (50mM) for cleavage of disulfide bonds.
iii. Renaturation is achived by reducinnnng the concentration of chaotropic
agents by dialysis
2. Polishing step
• It is the step designed to remove trace contaminantes and impurities to
obtains a biologically ative protein with safely profile suitable for
pharmaceutical use.