The document discusses purification of recombinant proteins using affinity tags. It describes immobilized metal affinity chromatography (IMAC) as a widely used method to purify recombinant proteins fused to tags like histidine, GST or MBP. The document outlines the steps involved, including gene amplification, cloning, expression in bacteria or yeast, and purification. It focuses on using histidine tags and nickel-chelate affinity chromatography, noting the advantages of tags for simplifying purification and detection of recombinant proteins.

1. Purification of
Recombinant
Proteins
Sumedha Bobade
Ph.D Scholar
Animal Biotechnology

2. Recombinant Protein
Purification
• Immobilized metal affinity chromatography (IMAC) It is widely used
method for purifying proteins of interest based on well developed
recombinant DNA and protein expression technologies. All proteins are
purifies using the method regardless of solubility of the proteins produced
in expression host cells.
• When the recombinant protein fused to peptide or protein tag such as
polyhistidine (His), glutathione –S-transferase (GST) ,Maltose binding
protein (MBP) or Step –tag II the properties of tag can be exploided for
purification purpose.

3. Steps to produce recombinant
protein
• 1.Amplification of gene of interest.
• 2.Insert into cloning vector.
• 3.Sub cloning into expression vector.
• 4.Transformation into protein expressing bacteria (E coli) or yeast.
• 5.Test for identification of recombinant protein.
• 6.Large scale production.
• 7.purification.

4. Expression and sample preparation
• Components of the expression system
• A protein expression system includes, among other things, a vector with an
appropriate promoter and other regulatory sequences, along with the gene encoding
the recombinant protein of interest. Vectors are available commercially for the
expression of recombinant proteins either fused to a tag or untagged. Such
expression vectors are designed with control regions to suit the specific host (for
example, E. coli versus mammalian cells) and type of expression needed.
• Choice of host
• The choice of host affects not only the expression of the protein but also the way in
which the product can be subsequently purified. In order to decide which host is
most suitable, the amount and the degree of purity of the product, as well as its
biological integrity and potential toxicity, should be considered. For example,
bacterial expression systems are not suitable if posttranslational modification is
required to produce a fully functional recombinant product. The location of product
within the host will affect the choice of methods for isolation and purification of the
product.

5. Expression and sample preparation
• Strain of E. coli for Protein Expression :
• For high-level protein production purposes, BL21(DE3) is an appropriate E. coli
strain. It has the advantage of being deficient in both lon and ompT proteases and it
is compatible with the T7 lacO promoter system. For eukaryotic proteins, it is often
important to use BL21(DE3) derivatives carrying additional tRNAs to overcome
the effects of codon bias. Historically, ampicillin has been the most commonly used
antibiotic-selection marker, but itis being replaced by carbenicillin, which is more
stable. Vectors encoding resistance to kanamycin or chloramphenicol are now
widely used as well.
• The difference between BL21 and BL21(DE3) competent E.coli cells?
• Both strains are B strains and thus both are deficient in Lon protease (cytoplasm)
and OmpT protease (outer membrane). Accordingly, B strains are generally
preferred for recombinant protein expression. The DE3 designation means that
respective strains contain the λDE3 lysogen that carries the gene for T7 RNA
polymerase under control of the lacUV5 promoter. IPTG is required to maximally
induce expression of the T7 RNA polymerase in order to express recombinant
genes cloned downstream of a T7 promoter.

6. Choice of vector
• The choice of vector family is largely governed by the host. Once the host
has been selected, many different vectors are available for consideration,
from simple expression vectors to those that contain specialized sequences
needed to secrete the recombinant proteins. In order to clone the gene of
interest, all engineered vectors have a selection of unique restriction sites
downstream of a transcription promoter sequence. Recent developments in
cloning technology provide increased flexibility in the choice of host and
vector systems, including options allowing the DNA sequence of interest to
be inserted into multiple types of expression vectors.
• The expression of a recombinant protein fused to a tag of known size and
biological function can greatly simplify subsequent purification and
detection (for expression method development and purification).

7. Choice of tag
• There are several affinity tags that can be used to simplify protein
purification. The choice of tag may depend on many different factors. The
most common tag, the histidine tag, is often a (histidine)6, but other
polyhistidine tags consisting of between four and 10 histidine residues have
been used. The latter provides for the strongest affinity for the
chromatography medium. Other important tags are the GST and MBP tags,
both of which are proteins, and Strep-tag II, which is a peptide optimized
for chromatography on Strep-Tactin™ based chromatography media.
• The rationale for the choice of an N-terminal hexahistidine is manifold.
First, an N-terminal tag ensures that the bacterial transcription and
translation machineries always encounter 5′ and N-terminal sequences that
are compatible with robust RNA synthesis and protein expression,
respectively. Second, oligohistidine-tagged proteins can be purified using a
relatively simple protocol using immobilized metal affinity
chromatography (IMAC) .

8. The histidine tag
• The DNA sequence specifying a string of six to nine histidine residues is frequently
used in vectors for production of recombinant proteins. The result is expression of a
recombinant protein with a 6xHis or poly-His-tag fused to its N- or C-terminus.
• Expressed His-tagged proteins can be purified and detected easily because the
string of histidine residues binds to several types of immobilized metal ions,
including nickel, cobalt and copper, under specific buffer conditions. In addition,
anti-His-tag antibodies are commercially available for use in assay methods
involving His-tagged proteins. In either case, the tag provides a means of
specifically purifying or detecting the recombinant protein without a protein-
specific antibody or probe.
• Protein purification
• As a chromatographic procedure, IMAC has the advantages of having strong,
specific binding, mild elution conditions and the ability to control selectivity by
including low concentrations of imidazole in chromatography buffers. There is a
broad array of common resins with slightly different binding capacities and binding
strengths, but all tolerate harsh cleaning procedures.

9. Various purification methods
• Affinity chromatography
• Based on Charge: Isoelectric Focusing
• Based on Hydrophobicity: Hydrophobic Interaction Chromatography
• Based on Size: size exclusion chromatography
• Immunoaffinity purification
• Fast Protein Liquid Chromatography (FPLC) Methods
• Reverse Phase HPLC (RP-HPLC)

10. Affinity chromatography using His-tag
• In this technique adding extra amino acid to the protein that give it a new
property which is bind very tightly to a particular substance.This binding
then can be use to isolate the protein away from other material in the cell
which do not bind this substance.This method is called as Affinity
chromatography using His-tag to purify protein.
• A his tag is a a string of (usually six) histidine residue when the gene is
cloned .
• Commercial plasmid vectors are available that already contain the codon
for this his-tag ,making the production of such protein relatively
straightforward.
• The presence of extra his-tag on the protein often does not interfere with
the ability of the protein to fold correctly and carry out its normal
function ,but it does make the protein bind very tightly to metal ion such
as Ni2+ .

11. Affinity chromatography
• A commercial resin is available that has Ni2+ ions attached to it ,so if this
resin is placed in a column and a crude extract of E.coli cells that
contain the His-tagged protein is passed down the column, the E. coli
will pass through the column but the protein with attached his tag will
bound to the column
• If a buffer containing a salt such as imidazole is subsequently passed
down the column, the bound his-tagged protein is now released from the
nickel ions, and can be collected in the buffer as it flows out of the
column.
•

12. Immobilized metal affinity
chromatography (IMAC)
• beaded agarose or magnetic particles can be derivatized with
chelating groups to immobilize the desired metal ions, which then
function as ligands for binding and purification of biomolecules of
interest. This basis for affinity purification is known as immobilized
metal affinity chromatography (IMAC). IMAC is a widely-used
method for rapidly purifying polyhistidine affinity-tagged proteins,
resulting in 100-fold enrichments in a single purification step.
• The chelators most commonly used as ligands for IMAC are
nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). Once IDA-
agarose or NTA-agarose resin is prepared, it can be "loaded" with
the desired divalent metal (e.g., Ni, Co, Cu, and Fe). Using nickel as
the example metal, the resulting affinity support is usually called Ni-
chelate, Ni-IDA or Ni-NTA resin. The particular metal and chelation
chemistry of a support determine its binding properties and
suitability for specific applications of IMAC.
• Affinity purification of His-tagged fusion proteins is the most common
application for metal-chelate supports in protein biology research.
Nickel or cobalt metals immobilized by NTA-chelation chemistry are

14. Advantages and disadvantages for using
tags in fusion proteins
• Advantages
• 1) Simple purification is possible using Affinity Chromatography . Improve
protein yield
• (2) Detection of the tag instead of the target protein moiety allows for a
generic detection method in, e.g., protein production platforms for
structural biology. Prevent proteolysis
• (3) Facilitate protein refolding : Some tags allow strong binding to
chromatography media in the presence of denaturants, making on column
refolding possible.
• (4) Protect the antigenicity of the fusion protein
• (5) Increase solubility: Solubility and stability can be improved.
• (6) Increased sensitivity.
• 7) Targeting information can be incorporated into a tag.
• 8) A marker for expression is provided.

15. Advantages and Disadvantages
• Disadvantages
• (1) A change in protein conformation
• (2) Lower protein yields
• (3) Inhibition of enzyme activity
• (4) Alteration in biological activity
• (5) Undesired flexibility in structural studies
• (6) cleavage/removing the fusion partner requires expensive protease
• (7) toxicity.
• 8) Tag may interfere with protein structure and affect folding and biological activity.
• 9) If tag needs to be removed, cleavage may not always be achieved at 100%, and
sometimes amino acids may be left.
• Untagged proteins
• Advantage: Tag removal is not necessary.
• Disadvantages
• Purification and detection not as simple.
• Problems with solubility and stability may be difficult to overcome, reducing potential
yield.

16. Ion Exchange Chromatography
• It is performed in a pH gradient in an electric field.
• The charged proteins migrates towards the anode or the cathode
according to the sign of their net charge until they reach the position in
the pH gradient where there net charges are zero.
• This pH value is isoelectric point (PI) of the substance,an exactly defined
physiochemical constant.
• Since the molecule is no longer charged it stays there the electric field
does not have any influence on it. The protein diffuse away it will gain
the net charge and applied electric field will cause it to migrate back to
its pI .this concentrating effect leads to the name focusing and makes the
method very useful for purification purpose

17. Ion Exchange Chromatography

18. Size Exclusion Chromatography: Gel
filtration chromatography - separation
by size• It is also known as gel filtration chromatography is a technique for
separating proteins and other biological macromolecules on the basis of
molecular size .
• The solid phase matrix consist of porous beads (100-250 μm) that are
packed into a column with a mobile liquid phase flowing through the
column. The mobile phase has access to both the volume inside the pores
and the volume external beads . The high porosity typically leads to a
total liquid volume of ˃ 95% of the packed column.
• Separation can be visualized as reversible portioning into the two liquid
volumes. Large molecules remain in the volume external to the bead
since they are unable to enter the pores.

19. Size Exclusion Chromatography:
Gel filtration chromatography -
separation by size

20. Chromatography on Hydroxyapatite:
Hydrophobic interaction chromatography (HIC)
• Hydrophobic interaction chromatography (HIC) separates molecules based
on their hydrophobicity. HIC is a useful separation technique for purifying
proteins while maintaining biological activity due to the use of conditions
and matrices that operate under less denaturing conditions.
• 2.The principle for protein adsorption to HIC media is complementary to
ion exchange and size exclusion chromatography. Sample molecules
containing hydrophobic and hydrophilic regions are applied to an HIC
column in a high-salt buffer. The salt in the buffer reduces the solvation of
sample solutes. As solvation decreases, hydrophobic regions that become
exposed are adsorbed by the media. The more hydrophobic the molecule,
the less salt is needed to promote binding. Usually a decreasing salt
gradient is used to elute samples from the column in order of increasing
hydrophobicity. Sample elution may also be assisted by the addition of
mild organic modifiers or detergents to the elution buffer.

21. Chromatography on Hydroxyapatite:
Hydrophobic interaction chromatography (HIC)
• 3.The method of hydrophobic interaction chromatography (HIC) is based
on the observation that protein molecules can interact with fully
hydrophobic adsorbents, and this interaction is dependent on the salt
concentration of the solution. Similarly to the salting-out of proteins where
the increasing salt concentration will lead to the aggregation and
precipitation of protein molecules via the rearrangement of their hydrate
shell, during HIC chromatography a high salt concentration (1-1.5 M
neutral salt) facilitates the interaction between the hydrophobic
chromatographic medium and the hydrophobic patches present on protein
molecules. During separation, the decrease in salt concentration will lead to
the elution of bound molecules.
• 4.Hydroxyapatite (HT/HTP) is a crystalline form of calcium phosphate
with the molecular formula Ca10(PO4)6(OH)2 . When the HT/HTP is
equilibrated with phosphate buffer it is then appears that positively
charged proteins interacts nonspecifically with the general negative
charged on the column produced by immobilized phosphate ions.

22. Chromatography on Hydroxyapatite:
Hydrophobic interaction chromatography (HIC)
• 5.The protein then can be eluted by increasing the phosphate
concentration ,addition of a salt such as sodium chloride or by the use of
Ca2+ or Mg 2+ , which complex with phosphate ions on
• Hydroxyapatite (HT/HTP) has achieved only on limited popularity as a
chromatographic material for the purification of proteins.
• 6.This is for a variety of reason including difficulties in predicting it . A
chromatographic behavior ,is relatively low capacity and the fact that its
handling properties are not ideal.

23. Affinity Precipitation Method
• Affinity chromatography is powerful protein purification technique ,that
exploits the specific interaction between a biological ligand ( eg.
Substrate , coenzyme, hormone, antibody or nucleic acid) or its synthetic
analog and its complementary binding site on protein.
• One of the variation of this technique is Affinity precipitation.
• There are two main approaches t oaffinity precipitation .The first is
called the bis-ligand or homobifunctional ligand approach .The ligand is
bifunctional bearing two identical ligand connected by a spacer arm.If
the spacer is long enough each ligand can bind to ligand binding site on a
different protein molecule .Oligomeric proteins can bind two or more bis-
ligand with the consequent formation of crosslinked lattices .
• The second approach to affinity precipitation differs from this in that
athe affinity ligand has two function one of which bind the target protein
and second to promote the precipitation of the aggregate.

24. Immunoaffinity purification
• The immunoaffinity chromatographic purification technique of
fusion protein with reference to human interleukin is described
• Interleukin 2 is joined to a small DNA sequence encoding a marker
peptide (That synthesized 8 amino acid ,Asp-Tyr-Lys-Asp-Asp-Asp-
Asp-Lys) produces a fusion protein in yeast (S.cerevisiae) .
• The marker peptide has a dual reduces the degradation of
interleukin 2 beside helping in its purification.(The eight amoino
acid marker peptide is commercially available under the brand
name Flag peptide).
• The fusion protein interleukin 2 joined to a marker peptide can be
purified by immunoaffinity chromatography.
• The specific monoclonal antibodies (MAb) against the marker
peptide are immobilized on polypropele support. These MAb
serves as the ligand with marker peptide antibodies and
selectively binds to fusion proteins tagged with marker peptide.
• However the remaining protein pass through the immunoaffinity
column. The immunopurified fusion protein can be elute later from
the column.

25. Immunoaffinity purification

26. Fast Protein Liquid
Chromatography (FPLC) Methods
• High performance liquid chromatography (HPCL) procedures exploits
column packaging with average diameter of as 5-40 μ However these
are used in high pressure system often with organic solvent and are
generally limited to rather low sample loading.
• FPLC provides a full range chromatography mode such as ion exchange,
chromatofocusing, gel filtration, hydrophobic interaction and reverse phase
based on particles with average diameter sizes in the same range as those
used for HPLC saperation.

27. Reverse Phase HPLC (RP-HPLC)
• This is well established technique for isolation ,analysis and structural
elucidation of peptides and proteins. It is used in protein isolation and
purification and have reached peak owing to recent development in high
affinity ion exchange and hydrophobic interaction support which are now
capable of equivalent levels of resolution to RP-HPLC
• The solvent gradient is generally superior to isocratic elution between
saperation is then achievable within reasonable time frame and peak
broadening of later elution peaks is reduced and thus sensitiveity
increased .In gradient elution RP-HPLC, proteins are retained essentially
according to their hydrophobic character, the retaintion mechanis can be
considered either as adsorption of the solute at the hydrophobic stationary
surface or as a partition between the mobile and stationary phase.

28. Manual purification techniques
• For small-scale purification of tagged proteins, a single affinity chromatography
step with a simple elution by a step gradient is usually sufficient. Manual
purification can be performed in batch or by using gravity-flow or spin columns, or
96-well plates.
• When a tagged protein is purified by a batch method, the protein sample is added to
a purification medium usually in a disposable plastic tube. The chromatography
medium is then washed and the tagged protein is eluted. The batch method is suited
to purification on a small scale.
• A tagged protein can also be purified by simply passing the protein sample through
a disposable
• column prepacked with an appropriate medium. There are columns especially
designed for use by gravity flow, for example, His GraviTrap for histidine-tagged
proteins. A 1 ml His GraviTrap column can purify approximately 40 mg of a
histidine-tagged protein. In addition, there are HiTrap columns suitable for use with
a syringe or peristaltic pump for histidine-, GST-, MBP-, and Strep-tag IIproteins,
(HisTrap, GSTrap™, MBPTrap™, and StrepTrap™ columns, respectively). In
general the binding capacity for a histidine-tagged protein using a HisTrap column
is at least 40 mg per ml of chromatography medium. HiTrap columns can also be
connected to AKTAdesign™ chromatography systems . Connections are easy to
make because HiTrap columns come with all necessary connectors included.

29. Protein characterization
• Characterizing the purified protein in some detail reduces the risk of
wasting resources on protein material of inadequate quality. It also provides
a means to ensure that different batches of the same protein have similar
properties. A simple, generic protein characterization protocol that allows
the experimentalist to judge whether the correct protein has been purified,
whether additional molecular species are present and to estimate the
approximate protein concentration are outlined below . Other
characterization methods that are very informative but not as widely
applied, such as mass spectrometry, static or dynamic light scattering, and
measuring protein thermal stability, are also described .

30. Protein characterization
• Inspection of gel filtration chromatogram
• If size exclusion chromatography was used as the last purification step, a
close look at the chromatogram is essential. Symmetric elution profiles are
characteristic of homogeneous proteins, whereas asymmetric profiles
reflect inhomogeneous, or partially aggregated, samples , or whether the
column itself is in poor condition. The elution profiles will also reveal the
primary oligomerization state. The presence of additional oligomerization
states may be of biological significance, or may be a sign of nonspecific
aggregation. If the protein elutes in the void volume of the chromatogram,
the protein is most likely forming large, nonspecific aggregates, which may
be an indication of improper folding and compromised activity. It is also of
value to analyze individual peaks by SDS-PAGE or mass spectrometry to
analyze the protein in each peak.

31. Protein characterization
• SDS-PAGE analysis
• After protein purification, samples should be resolved by denaturing
SDS-PAGE. If stained with a dye such as Coomassie brilliant blue,
the intensity of the bands will usually be proportional to the amount
of protein. This allows the purity of the sample to be estimated and
whether the purified protein is of the expected size.
• UV absorption spectroscopy
• To quantify the amount and concentration of purified protein, the
simplest and most common method is the Bradford assay, which
measures the binding of Coomassie brilliant blue to the protein. As
some proteins bind the dye anomalously, it is also useful to measure
the UV absorption at A280 and calculate the concentration of the
protein by using the predicted molar extinction coefficient at A280 By
taking a UV absorption spectrum, it is also possible to uncover
contamination with DNA or RNA, or reveal common copurifying
cofactors (for example, NAD, FAD, heme).

32. Storing purified protein
• Aliquots of the protein to be stored should be placed in thin-walled PCR
plastic tubes, frozen in liquid nitrogen and stored at −80 °C. Small aliquots
should be frozen to avoid damaging freeze-thaw cycles, and aliquots should
be thawed on ice. Concentrated proteins (for example, >1 mg/ml) tend to
be more stable to freeze-thaw cycles.
• Proteins are usually concentrated using centrifuge-driven filter devices with
adequate molecular weight size cutoffs. Care should be taken during
centrifugation to avoid local over-concentration and irreversible
precipitation or aggregation of the protein on the filtration membrane.
• It is advisable to explore the stability of the protein to concentration and
freeze-thaw cycles before processing the entire batch. The frozen and
thawed sample should be compared with protein that was not frozen for
biochemical activity, visible precipitation, changes in physical properties
(for example, dynamic light scattering or gel filtration profile) or
crystallization characteristics. In our collective experience, relatively few
proteins are irreversibly inactivated by one freeze-thaw cycle. In those rare
instances, the protein can be stored at 4 °C for short periods of time, at −20
°C in high concentrations of glycerol, or as an ammonium sulfate
suspension

33. References
• Graslund et al., (2008) Protein production and purification. Nat Methods,
5(2):135–146.
• Guzman LM, Belin D, Carson MJ, Beckwith J. (1995) Tight regulation,
modulation, and high-level expression by vectors containing the arabinose
pBAD promoter. J. Bacteriol.; 177:4121– 4130.
• Uhlen M, Forsberg G, Moks T, Hartmanis M, Nilsson B. (1992) Fusion
proteins in biotechnology. Curr. Opin. Biotechnol.; 3:363–369.