Isolation & production of r-proteins

1. Strategies for commercial
production , isolation/purification
of recombinant protein.

2. What is recombinant protein?
Recombinant protein is a manipulated form of protein, which is generated in
various ways to produce large quantities of protein modify gene sequence and
manufacture useful and commercial products.

3. Production of recombinant protein
• Transcription and translation steps belong to the recombinant
protein expression systems.
• To make recombinant proteins, the gene is isolated and cloned into
an expression vector. Generating a r protein requires the protein
expression system, protein purification system, protein identification
systems.
At DNA level :- 1. finding the protein of interest
Sequence has to be determined from wild type protein
2. Deletion mutants:- AA can be deleted from the sequence, either
individually or in groups
3. Point mutants:- at any position in the sequence an amino acid can
be replaced by another to generate a mutant protein
(this mutant protein is expected to have little or no
biological activity ,because histadine 119 is important for
that activity )

4. Suitable expression system
1. Bacterial expression system.
2. Yeast expression system.
3. Insect/Baculovirus expression system.
4. mammalian expression system.
Through r DNA technology, a large quantity of
proteins can be produced. This involves
inserting the desired protein gene into an
expression vector which must contain promoter
so that the protein can be expressed

5. Stategies
• Rational designing of proteins by site
directed mutagenesis
• Directed evolution of proteins by random
mutagenesis
Before engineering characteristics of wild type
protein must be determined (variety of
analytical methods)
Production of mutants protein to assess elements
that are necessary for its function

6. Pre conditions
• Working knowledge of protein
• Locating a desired protein
• Clone and express
• The design and construction of new proteins or
enzymes with novel or desired function, through
the modification of amino acid sequence

7. • There are basically two methods for producing
recombinant proteins.
• One is molecular cloning, a laboratory method used
to make recombinant DNA.
• The other method is the polymerase chain reaction
used to proceed the replication of any specific DNA
sequence selected.
Cloning process :- 1. G
ene of interest is cut out with restriction enzymes
2. Host plasmid is cut with same RE
3. Gene is inserted into plasmid and ligated with ligase.
4. New (engineered) plasmid inserted in to
bacterium(transformed)

8. Hepatitis B vaccine
• The gene encoding for Hepatitis B surface antigen (HBsAg) has been identified.
• The HBsAg vaccine as a subunit vaccine, is produced by cloning HBsAg gene in yeast
cells

9. Isolation of recombinant protein
• Recombinant protein are isolated from very complex
protein mixture present in producing cell.
• The isolation process involves in general four
mutually interconnected stages:-
1. Release of recombinant protein from the cellular
environment.
2. preparation of the specimen for separation
3. Separation.
4. Qualitative and quantitative analysis of the
preparation.

10. • => in addition to the protein of interest, several thousand
other protein with different properties are present in the
extract, along with nucleic acid (DNA and RNA),
polysaccharide, lipid, and small molecules.
• the challenge is therefore, is to separate the protein of
interest from cell of other components in the cell,
especially the unwanted contaminating proteins with
reasonable efficiency, speed, yield, and purity while
retaining the biological activity and chemical integrity of
polypeptide.
• Extraction process
• 1. Cell lysis (osmotic shock) -> protein source -> E.coli periplasm intracellular
protein
• 2. Enzymatic digestion -> protein source -> intracellular protein /periplasm
• 3. Ultrasonication -> cell suspension -> inclusion bodies
• 4. Manton-gaulin homogeniser-> cell suspension large scale only.
Note :- If lysates are to viscous to handle which caused by the presence of high
concentration of host nucleic acid give DNAse and RNAse.

11. By exploiting the difference in properties between the protein of interest and
other proteins in the mixture, rational series of fractions steps can be
designed. These properties include:
• Size
• Shape
• Charge
• Isoelectric point
• Charge distribution
• Hydrophobicity
• Solubility
• Density
• Ligand binding
• Metal binding
• Reversible association
• Post translational modification
• Specific sequence or structure
Biological source : take a tissue/cell type that contain large amounts of the
target protein.
Lyse cell : separate cell homogenate into fractions

12. Strategies of purification of r-
proteins:
CAPTURE STEP
• Isolate, concentrate,
and stabilize the target
product.
• Effectiveness assessed
based on
chromatography speed
POLISHING STEP
• Includes a capture
step and one or more
polishing steps.
• Flow rates and load
volumes of the
polishing steps may
be decreased in order
to improve
resolution.
Removes most of the bulk
impurities (other proteins,
nucleic acids, endotoxins,
viruses)
INTERMEDIATE
STEP

13. Performance parameters
1. Resolution
2. Capacity
3. Speed
4. recovery
Increasing the number of purification steps will often
decrease the overall protein recovery

14. CAPTURE STEP
• Definition: Initial purification of the
target molecule from the source
material.
• Goal: Rapid isolation, stabilization,
and concentration.
• Removal of critical contaminants to
obtain high level of purification.
• IEX or AC
• Target protein isolated from critical
impurities (proteases and
glycosidases).
• Maximizing capacity and/or
speed will be at the expense of
some resolution.

15. Intermediate step
• Definition: Further removal
of bulk contaminants.
• Goal: Purification and
concentration.
• Eradication of Impurities like
other proteins, nucleic acids,
endotoxins, and viruses.
• The requirements for
resolution will depend on the
properties of the sample
produced from the capture step
and the purity requirements for
the final product

16. Polishing step
• Definition: Final removal of trace
contaminants and adjustment of pH,
salts, or additives for use or storage.
• Goal: End product of required high-
level purity.
• Trace impurities removed (e.g.,
endotoxins, nucleic acids, or viruses),
and closely related proteins such as
microheterogeneous structural variants.
• To achieve enough resolution it may be
necessary to sacrifice sample load
(overload may decrease purity) and
recovery by narrow peak fractionation.
• Recovery of product in buffer
conditions.

17. Selection and combination of
purification methods

18. AC: Single-step purification of affinity-tagged proteins using AC may give sufficient purity for some applications.
AC-GF: Complementing an AC step with polishing by GF is very common, and can often be used for generic setups
when regularly purifying multiple proteins.
AC-IEX-GF: For high-purity requirements in affinity-tagged protein purification, AC-IEX-GF is a powerful and
convenient combination.
Untagged proteins can usually be sufficiently purified by combining purification methods that separate on the basis
of different physicochemical characteristics of the proteins (orthogonal methods).
IEX-HIC-GF: The combination IEX-HIC-GF is a very often used three-step purification because the high-salt
conditions after the first step can simply be adjusted with additional salt for HIC purification and followed by GF for
polishing and salt removal.
HIC-IEX-GF: If ammonium sulfate precipitation has been performed, the combination HIC-IEX-GF is suitable
because HIC requires high-salt conditions for binding and gives elution in a relatively low salt concentration in a
significantly smaller volume. Dilution or desalting can then be used to remove remaining salt, so that the sample can
be bound to an IEX column.

19. Purification of untagged r-
protein by column
chromatography:
• Various chromatographic techniques developed
based on protein molecules size, shape, overall
charge, presence of surface hydrophobic groups
and ability to bind various ligands.
• Chromatographic Techniques commonly used and
their basis of separation are as follows:
• Ion exchange – protein surface charge at given pH
• Hydrophobic Interaction Chromatography –
surface hydrophobicity of proteins
• Gel filtration chromatography – mass or
size/shape of proteins
• Chromatofocussing – isoelectric points
• Affinity chromatography – biospecific interaction
between protein and a ligand

20. Chr media The particle size of the medium strongly
affects efficiency and flow resistance.
In the polishing stage, focus is put on high
purity, which can be obtained with
chromatography media with high
efficiency, that is, small beads.

21. Gel filtration
• Smaller solutes retain on the
column longer
• Therefore, retention time is
inversely proportional to the
size of the solute
Dextran
(=sephadex)
Polyacrylamide
Agarose
(=sepahrose)
0.15-1.0 M salt buffer to prevent interaction of the protein with support
matrix.
Unlike others, protein doesn’t bind stationary phase during the process.
Resolution depends on smallest possible volume of sample.

22. • Also, used to determine molecular wt. of a protein.
Vo = Void Volume (solvent between beads)
Vt = Total Volume (pi r 2 into length)
Ve = Elution Volume
Shape
~ long rod shaped proteins elute at apparent molecular weights greater than their actual
molecular weights.
DESALTING – removing of salts from protein solutions or to carry out buffer exchanges.
For e.g. Desalting columns much faster than dialysis.
Spins columns.

23. Purification of tagged r-protein by
column chromatography:
• Undergoes single step protein purification (capture step),
when suitable ligand is available for protein of interest
followed by second chromatographic step (polishing step) to
remove remaining impurities.
• Affinity chromatography - on the basis of reversible
interaction between protein and a specific ligand attached to a
chromatographic matrix
• Unbound material washed away and bound target protein is
by desorption.
• Affinity tags - highly efficient tools for protein purification.
• Inteins tags - to purify any r-proteins of interest in column
chromatography. It excise itself and rejoin exteins with a
peptide bond. Purification of protein in column
chromatography is without the use of protease to remove the
tag (self-cleavable proteases).

24. ION EXCHANGE CHROMATOGRAPHY
Chromatography is the separation of a mixture of compounds into its individual components
based on their relative interactions with an inert matrix.
Ion exchange chromatography is a process that allows the separation of ions and polar
molecules based on their affinity to ion exchangers.
• This technique is extremely useful in the separation of charge compounds like proteins
differing by only one charged amino acid.
• In ion exchange chromatography technique one can choose whether to bind the
substance of interest and allow the contamination to pass through the column and vice
versa.
PRINCIPLE
• Ion exchange chromatography relies on the attraction between oppositely charged
stationary phase,known as an ion exchanger and analyte.
• The ion exchanger consists of a inert support medium coupled covalently to
positive(anion exchanger)or negative(cation exchanger) functional groups.
• To these covalently bound fuctional groups the oppositely charged ions are bounded
(mobile counter ion),which will be exchanged with like charge ions in the sample having
charge magnitude more than the ions bounded to the matrix.
• Thus if anion exchange chromatography is performed,negatively charged sample
components will interact more with the stationary phase and will be exchanged for like
charged ions already bounded to the matrix.

26. AFFINITY CHROMATOGRAPHY
Affinity chromatography is a type of liquid chromatography for the
separation,purification or specific analysis of sample components.
It utilizes the reversible biological interaction or molecular recognition called
affinity which refers to the attracting forced exerted in different degrees between
atoms which cause them to remain in combination. Example: Enzyme with and
inhibitor, antigen with an antibody etc.
PRINCIPLE
• The stationary phase consists of a support system, on which the
substrate(ligand) is bound covalently, in such a way that the reactive groups
that are essential for binding of the target molecule are exposed.
• As the crude mixture of the substances is passed through the chromatography
column, substances with binding site for the immobilized substrate bind to the
stationary phase,while other substances is eluted in the void volume of the
column.
• Once the other substances are eluted,the bound target molecules can be eluted
by methods such as including a competing ligand in the mobile phase or
changing the pH, ionic strength or polarity conditions.

27. • The purity of products obtained in that way may be
adequate for basic research, but additional
purification steps are often required:
(a)Multimeric forms and/or fragments of the
recombinant protein produced by incomplete
translation are also adsorbed specifically.
(b)Small amounts of contaminants are adsorbed non-
specifically; and
(c)Losses of the ligand from the column can occur if
harsh conditions of pH are required for elution.

28. MECHANISM OF AFFINITY BINDING

29. Hydrophobic interaction
chromatography
Hydrophobic interaction chromatography is the separation
technique that separates molecules on the basis of their degree of
hydrophobicity.
PRINCIPLE
• The principle of hydrophobic interaction chromatography is based on
the interaction between two molecules with hydrophobic groups.
• Here, the stationary phase is solid support applied with both
hydrophobic and hydrophilic groups.
• The solvent molecules containing hydrophobic regions interact with
the hydrophobic groups, thus separating them from the molecules
with hydrophilic groups.
• The interaction is then reversed by applying an elution solution with
decreasing salt gradient, which causes the molecules with
hydrophobic groups to be separated from the stationary phase.

30. (A)Highly ordered water shells surround the hydrophobic surfaces of ligand and proteins.
Hydrophobic substances are forced to merge to minimize the total area of such shells[maximize
entropy].Salts enhance the hydrophobic interaction.
(B)The equilibrium of the hydrophobic interaction is controlled predominantly by the salt
concentration.

31. REVERSE PHASE CHROMATOGRAPHY
Reverse-phase chromatography is a liquid chromatography technique where
the separation of molecules is achieved through hydrophobic interaction between
the liquid mobile phase and the stationary phase.
PRINCIPLE
• The principle of reverse phase chromatography is based on the interaction
between two molecules with hydrophobic groups.
• Here, the stationary phase is solid support applied with both hydrophobic
and hydrophilic groups.
• The solvent molecules containing hydrophobic regions interact with the
hydrophobic groups, thus separating them from the molecules with
hydrophilic groups.
• The interaction is then reversed by applying an elution solution with
decreasing salt gradient, which causes the molecules with hydrophobic
groups to be separated from the stationary phase.

32. DIFFERENCE

33. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
High-performance liquid chromatography is a modified form of column
chromatography where the components of a mixture are separated on the basis of
their affinity with the stationary phase.
PRINCIPLE
• This technique is based on the principle of differential adsorption where
different molecules in a mixture have a varying degree of interactions with
the absorbent present on the stationary phase.
• The molecules having higher affinity remain adsorbed for a longer time
decreasing their speed of movement through the column.
• However, the molecules with lower affinity move with a faster movement,
thus allowing the molecules to be separated in different fractions.
• This process is slightly different from the column chromatography as in this
case; the solvent is forced under high pressures of up to 400 atmospheres
instead of allowing it to drip down under gravity.
•

35. REFERENCES
• https://books.google.co.in/books?hl=en&lr=&id=CN53kDLCOjAC&oi=fnd
&pg=PR5&dq=recombinant+proteins+purification&ots=RQPM95z49L&sig
=smJfh2qK9rkykriqQObYk0WK3x8#v=onepage&q=recombinant%20prote
ins%20purification&f=false
• https://www.researchgate.net/publication/289525226_Chromatography_-
_the_essence_of_bioanalysis
• https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201100015
• https://www.sinobiological.com/resource/protein-review/protein-
purification