BIOCHEMISTRY LEC

PROTEIN ISOLATION, PURIFICATION AND CHARACTERIZATION

MODULE 6
One of the biggest challenges in studying the detailed structure and properties of proteins is their
isolation and purification from cells and tissues with a highly complex composition and organization. Latest
refinements in the technology of protein purification and characterization have made this process easier and
quicker for researchers in this field. The extraction and isolation of proteins from cells and tissues is the first
important step towards the detailed study of proteins. Proteins are extracted from cells and tissues by
suspending them in the appropriate buffer and making a crude homogenate which can then be subjected to a
series of subcellular or differential centrifugation steps to remove cell debris and separate the various cell
fractions namely nuclear, mitochondrial, and microsomal fractions based on their sedimentation behavior when
subjected to increasing g forces (forces of gravity) in a centrifuge. The fraction which contains the protein of
interest is then chosen and the crude protein is salted out using ammonium sulfate, a common reagent used
to precipitate proteins from solution. The protein of interest will precipitate at specific percentage ammonium
sulfate saturation and can be localized in a particular fraction by following its activity. When localized, the
fraction is then dialyzed to remove excess ammonium sulfate, dried, and taken up in the appropriate buffer. It
is then ready for further purification and characterization steps which can involve a combination of high
performance liquid chromatographic and electrophoretic techniques. Once a pure sample of the protein is
obtained, it can now be subjected to other more sophisticated techniques such as mass spectrometry, protein
sequencing or X-ray diffraction techniques for further study of its structure and function.

Proteins are extracted, isolated, and purified from cells or tissues. As a summary:

1. The starting sample is suspended in the appropriate buffer and homogenizing it in a Potter-Elvehjem
tissue homogenizer or a similar device on ice at 0-4 oC until a crude homogenate is obtained.
2. Proteins are isolated from the homogenate by centrifugation and salting out. This protein isolates are
non-specific and does not discriminate the target protein from all other proteins in the sample.
 Subcellular or Differential centrifugation in a refrigerated centrifuge for separating proteins from
a solution according to their size, shape, density, viscosity of the medium and speed of the rotor
or force of gravity.
 The chosen fraction contains many other molecules aside from protein, thus proteins are salted
out or precipitated using different percentage saturation of ammonium sulfate, a common
reagent used to precipitate proteins out from solution. When localized, the fraction is then
dialyzed to remove excess ammonium sulfate, dried, and taken up in the appropriate buffer.
3. This fraction only contains proteins but it is a mixture of target protein and other proteins with similar
electrostatic characteristics. Further purification and characterization is done through chromatography
and electrophoresis.
4. Once a pure sample of the protein is obtained, it can bow be subjected to more sophisticated
techniques such as spectrometry, protein sequencing or X-ray diffraction techniques for further study
of its structure and function.

Different Protein Purification Methods based on:

 Charge : ion-exhange chromatography, isoelectric focusing

 Size : dialysis, gel filtration chromatography, gel electrophoresis, sedimentation velocity
 Solubility: Salting out
 Hydrophobicity : Reverse phase chromatography
 Specific binding : affinity chromatography
 Complexes : Immune precipitation (± crosslinking)
 Density : sedimentation equilibrium
CENTRIFUGATION

Centrifugation is used to separate particles of macromolecules such as cells, sub-cellular components,

proteins, and nucleic acids. It separates particles based on size, shape and density.

PRINCIPLES OF CENTRIFUGATION

 A centrifuge is a device for separating particles from a solution according to their size, shape, density,
viscosity of the medium and rotor speed.
 In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles
that are higher than it float to the top.
 The greater the difference in density, the faster they move. If there is no difference in density
(isopyknic conditions), the particles stay steady.
 To take advantage of even tiny differences in density to separate various particles in a solution, gravity
can be replaced with the much more powerful “centrifugal force” provided bu a centrifuge.

Figure 1. Differential Sedimentation of subcellular organelles

Density Gradient Centrifugation

 One of the most common and efficient type of centrifugation

 A gradient of sucrose (highest to lowest density if layered from bottom to top) is placed in the
centrifuge tube
 Homogenized samples are placed at the top of the sucrose gradient
 During centrifugation, the particles travel through the gradient until they reach the layer that matches
their density
 The target fraction is removed and subject to further analyses.

Figure 1. Density gradient centrifugation

 PRINCIPLE OF DENSITY GRADIENT CENTRIFUGATION

Differential Centrifuge

 The most common type of centrifuge

 It involves multiple centrifugation. At each round, a pellet is formed and the supernatant is transferred
to another tube.
 The supernatant will be centrifuged until another pellet is formed.

ADDITIONAL NOTES

 Centrifugation is a technique of separating substances which involves the application of centrifugal

force.
 The particles are separated from a solution according to their size, shape, density, the viscosity of the
medium and rotor speed.

Principle of Centrifugation

 In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles
that are lighter than it floats to the top.
 The greater the difference in density, the faster they move. If there is no difference in density
(isopycnic conditions), the particles stay steady.
 To take advantage of even tiny differences in density to separate various particles in a solution, gravity
can be replaced with the much more powerful “centrifugal force” provided by a centrifuge.
 A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a
circle), applying a potentially strong force perpendicular to the axis of spin (outward).
 The centrifuge works using the sedimentation principle, where the centripetal acceleration causes
denser substances and particles to move outward in the radial direction.
 At the same time, objects that are less dense are displaced and move to the center.
 In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to
settle to the bottom of the tube, while low- density substances rise to the top.
LOW-SPEED CENTRIFUGE

1) Most laboratories have a standard low-speed centrifuge used for routine sedimentation of heavy
particles
2) The low-speed centrifuge has a maximum speed of 4000-5000rpm
3) These instruments usually operate at room temperatures with no means of temperature control.
4) Two types of rotors are used in it,
 Fixed angle
 Swinging bucket.
5) It is used for sedimentation of red blood cells until the particles are tightly packed into a pellet and
supernatant is separated by decantation.

HIGH-SPEED CENTRIFUGES

1. High-speed centrifuges are used in more sophisticated biochemical applications, higher speeds and
temperature control of the rotor chamber are essential.
2. The high-speed centrifuge has a maximum speed of 15,000 – 20,000 RPM
3. The operator of this instrument can carefully control speed and temperature which is required for
sensitive biological samples.
4. Three types of rotors are available for high-speed centrifugation-
 Fixed angle
 Swinging bucket
 Vertical rotors

ULTRACENTRIFUGES

1. It is the most sophisticated instrument.

2. Ultracentrifuge has a maximum speed of 65,000 RPM (100,000’s x g).
3. Intense heat is generated due to high speed thus the spinning chambers must be refrigerated and kept
at a high vacuum.
4. It is used for both preparative work and analytical work.

Types of Centrifugation

1. Differential Pelleting (differential centrifugation)

 It is the most common type of centrifugation employed.
 Tissue such as the liver is homogenized at 32 degrees in a sucrose solution that contains buffer.
 The homogenate is then placed in a centrifuge and spun at constant centrifugal force at a constant
temperature.
 After some time a sediment forms at the bottom of a centrifuge called pellet and an overlying
solution called supernatant.
 The overlying solution is then placed in another centrifuge tube which is then rotated at higher
speeds in progressing steps.
2. Density Gradient Centrifugation
 This type of centrifugation is mainly used to purify viruses, ribosomes, membranes, etc.
 A sucrose density gradient is created by gently overlaying lower concentrations of sucrose on higher
concentrations in centrifuge tubes
 The particles of interest are placed on top of the gradient and centrifuge in ultracentrifuges.
 The particles travel through the gradient until they reach a point at which their density matches the
density of surrounding sucrose.
 The fraction is removed and analyzed.
 3. Rate-Zonal Density-Gradient Centrifugation
 Zonal centrifugation is also known as band or gradient centrifugation
 It relies on the concept of sedimentation coefficient (i.e. movement of sediment through the
liquid medium)
 In this technique, a density gradient is created in a test tube with sucrose and high density at the
bottom.
 The sample of protein is placed on the top of the gradient and then centrifuged.
 With centrifugation, faster-sedimenting particles in sample move ahead of slower ones i.e.
sample separated as zones in the gradient.
 The protein sediment according to their sedimentation coefficient and the fractions are collected
by creating a hole at the bottom of the tube.
4. Isopynic Centrifugation
 The sample is loaded into the tube with the gradient-forming solution (on top of or below pre-formed
gradient, or mixed in with self-forming gradient)
 The solution of the biological sample and cesium salt is uniformly distributed in a centrifuge tube and
rotated in an ultracentrifuge.
 Under the influence of centrifugal force, the cesium salts redistribute to form a density gradient from
top to bottom.
 Particles move to point where their buoyant density equals that part of gradient and form bands. This
is to say the sample molecules move to the region where their density equals the density of gradient.
 It is a “true” equilibrium procedure since depends on bouyant densities, not velocities

Eg: CsCl, NaI gradients for macromolecules and nucleotides – “self-forming” gradients under centrifugal force.

Applications of Centrifugation

 To separate two miscible substances

 To analyze the hydrodynamic properties of macromolecules
 Purification of mammalian cells
 Fractionation of subcellular organelles (including membranes/membrane fractions) Fractionation of
membrane vesicles
 Separating chalk powder from water
 Removing fat from milk to produce skimmed milk
 Separating particles from an air-flow using cyclonic separation
 The clarification and stabilization of wine
 Separation of urine components and blood components in forensic and research laboratories
 Aids in the separation of proteins using purification techniques such as salting out, e.g. ammonium
sulfate precipitation.

SALTING OUT

 Precipitation
 Based on solubility in various percentage of saturations of ammonium sulfate (NH4)2SO4
 Non-specific purification method
 Decreases the solubility of proteins by adding a high ionic solution. When the solubility decreases,
proteins start to precipitate.
 The opposite of salting in, wherein the solubility is increased.
AMMONIUM SULFATE “CUTS”

0-20%
21-40% DIALYSIS TO REMOVE
41-60% “CHOICE CUT” AMMONIUM SULFATE
61-80% AND CONCENTRATE THE
81-100% PROTEIN

Chromatography

Chromatography separates one protein from another based on the difference of different parameters
such as their size (size-exclusion chromatography), charge (ion-exchange chromatography), hydrophobicity
(hydrophobic interaction chromatography), or ability to bind a specific ligand (affinity chromatography)

Terms:

 Mobile phase - The liquid or gas (the solvent) that moves through the column
 Stationary phase- substance which the mobile phase passes through.
 Eluent- the fluid is added into the column
 Eluate- the fluid that exits the column and collected in the flask

Figure 1. Eluate, Eluent, Elution

Types of Chromatography

 Ion-exchange Chromatography
o Proteins interact with the stationary phase by charge-charge interactions
o At a given pH, proteins with a net positive charge will tightly bind to negatively charged beads
with functional groups such as carboxylates or sulfates (cation exchangers). Similarly, proteins
with a net negative charge will bind to positively charged beads, typically tertiary or quaternary
amines (anion exchangers).
o Nonadherent proteins passes through the matrix and eventually, washed away.
o The ionic strength of the mobile phase is gradually increased (consequently weakening charge-
charge interactions) to selectively displace bound proteins. Bound proteins then are selectively
displaced by gradually raising the ionic strength of the mobile phase, thereby weakening charge-
charge interactions
o Proteins elute in decreasing order of the strength of their interactions with the stationary phase
(stronger affinity, slower movement), thus, the net charge of a protein at the pH used determines
the rate of their movement through the column. A protein with a more negative net charge will
move faster and eluate earlier with cation exchangers.
 Figure 2. Ion-Exchange Chromatography

 Size exclusion Chromatography

o Separates proteins according to their size (stokes radii). Stokes radius is a measure of the
volume occupied by a protein in a free solution.
o Also called as gel filtration chromatography
o Protein with too large Stokes radii to enter the pores of the stationary phase will remain in the
flowing mobile phase, therefore, will be eluted first.
o Proteins emerge from the gel filtration column in descending order of their Stokes radii.

Figure 3. Gel Filtration Column. Large molecules move through the column faster than smaller
molecules

 High Performance Liquid Chromatography

o Separates compounds in a liquid mixture.
o The liquid sample is injected to the solvent (mobile phase) pumped at steady rates through the
column packed with a separation medium (stationary phase)
 o Separation of the components is based on their partition to the stationary and mobile phase, by
a process of differential migration as they flow through the column.

Figure 4.As bands emerge from the column, flow carries them to one or more detectors which deliver a
voltage response as a function of time. This is called a chromatogram.

Figure 5. Basic Components of HPLC

 Affinity Chromatography
o Based on the high selectivity of proteins to their ligands.
o Proteins that interact with immobilized ligands adhere.
o Bound proteins are eventually washed out by either by competition with free, soluble ligand
or, less selectively, by disrupting protein-ligand interactions using urea, guanidine
hydrochloride, mildly acidic pH, or high salt concentrations

Figure 5. Protein Affinity Chromatography

 Figure 6. Antibody- Affinity Chromatography

Electrophoresis

o The process of separating nucleic acids and proteins based on their size and electric charge.
o A widely used technique to determine the purity of proteins and subunit components
o This method can also help diagnose certain conditions. For instance, the amount of protein in the blood
or urine is measured through electrophoresis, and compare it to established normal values

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

o SDS is also commonly used in saponification method. It is commonly found as an ingredient in

shampoo, soaps and cosmetics (lauryl sulfate).
o An anionic detergent - it has a net negative charge.
o SDS is a strong denaturing agent. The negative charges of the SDS denature complex structure of
proteins and are strongly attracted to the positively charged electrode
o SDS binds to the protein backbone at a constant ratio.
o Polyacrylamide Gel provides a mesh-like matrix that separates proteins based their sizes. This matrix
allows smaller molecules to travel faster than the larger molecules.
o Molecular mass is expressed in Dalton (Da). Macromolecules are commonly expresses as kilodalton
(kDa)
Figure 1. SDS Page
 Figure 3. SDS page bands of Serum Protein

Figure 2. SDS Page of Serum Protein

Figure 4. Easy way to remember serum protein electrophoresis
Isoelectric Focusing (IEF) Electrophoresis

 Separates proteins based on their isoelectric points

 Proteins have a positive charge when at a pH below their isoelectric point, therefore, will move towards
the cathode. Likewise, proteins above the isoelectric point will have a negative charge and will move
towards the anode. The charge becomes zero at the isoelectric point and will no longer migrate.
 IEF is performed in a pH gradient
 Ionic buffers called Ampholytes are used to create pH gradient in IEF.
 Figure 5. Movement of amino acids below and above their isoelectric point

Western Blotting

Blotting is a technique wherein the isolated nucleic acids or proteins are transferred into a membrane.

o Southern blot: used to transfer DNA

o Northern blot: used to transfer RNA
o Western blot: used to transfer proteins
 Western blot is used to detect specific proteins in a sample
 Antibody of the target protein is used in this technique
 It also referred to as immunoblot

Figure 6. Detection of proteins using western blot

PROTEIN SEQUENCING

PURE PROTEINS ARE FURTHER ANALYZED BY:

1) Mass Spectrometry - Molecular Weight

2) Protein Sequencing (De Novo Analysis, Edman Degradation, Enzyme Restriction) - Primary Structure
3) X-Ray Diffraction - Secondary and Tertiary Structures

Mass Spectrometry (MS)

 Mass Spectrometry is an analytical technique that separates the components of a sample according to
their mass and charges. Therefore, charged peptides are separated based on their mass/charge (m/z)
 The instrument used in MS is called mass spectrometer
 Tandem Mass Spectrometry (MS/MS) further breaks the peptides into fragment ions. The mass of each
piece is then measured.

Figure 1. Sequence of events defining a contemporary mass spectrometry (MS)-based proteomics

experiment

Figure 2. Pipeline involved in Peptide Sequencing using Tandem Mass Spectrometry

Protein Sequencing

1) De Novo Sequencing
 The analytical process that gives the amino acid sequence from the tandem mass spectrum (MS/MS)
 Sequencing of proteins not found in the sequence database requires de novo sequencing
 The masses of the molecular fragments will give 'mass spectrum" by which the sequence can be
derived

2) Edman Degradation
 The process of determining the sequence of a peptide by cleaving the
N-terminal with the reagent PITC (phenylisothiocyanate)
I. PITC (phenylisothiocyanate) is added to the N- Terminal of a
peptide/ protein under mild alkaline condition.
II. The PITC conjugated N- terminal amino acid is cleavage as a
thiazolinone derivative under acidic conditions.
III. The PITC-AA is converted to the more stable PTH-AA
(phenylthiohydantoin amino acid derivative), and identified by
chromatography. Meanwhile, the cycle of PITC reaction is repeated
for identification of the next amino acid.

Figure 3. Edman Degradation

3) Enzyme Restriction
Reagent Cleavage site
Chemical cleavage
Cyanogen bromide Carboxyl side of methionine residues
o- Iodosobenzoate Carboxyl of tryptophan residues
Hydroxylamine Asparagine-glycine bonds
2-Nitro-5-thlocyanbenzoate Amino side of cysteine residues
Enzymatic cleavage
Trypsin Carboxyl side of lysine and arginine residues
Clostripain Carboxyl of arginine residues
Staphylococcal protease Carboxyl side of aspartate and glutamate
residues (glutamate only under certain
conditions)
Thrombin Carboxyl side of arginine
Chymotrypsin Carboxyl side of tyrosine, tryptophan,
phenylalanine, leucine, and methionine
Carboxypeptidase A Amino side of C- terminal amino acid (not
arginine, lysine or proline)

THE SPECIFICITY OF SOME COMMON METHODS FOR FRAGMENTING POLYPEPTIDE CHAINS

Reagent (biological source) Cleavage points

Trypsin (bovine pancreas) Lys, Arg (C)
Submaxillary prostease (mouse submaxillary Arg (C)
gland)
Chymotrypsin (bovine pancreas) Phe, Trp, Tyr (C)
Staphylococcus aureus V8 protease (bacterium S. Asp, Glu (C)
aureus)
 Asp-N-protease (bacterium Pseudomonas fragi) Asp, Glu (N)
Pepsin (procine stomach) Leu, Phe, Trp, Tyr (N)
Endiproteinase Lys C (bacterium Lysobacter Lys (C)
enzymogenes)
Cyanogen bromide Met (C)
*All reagents except cyanogen bromide are proteases. All are available from commercial sources.
*Residues furnishing the primary recognition point for the protease or reagent; peptide bond cleavage
occurs on either the carbonyl (C) or the amino (N) side of the indicated amino acid residues.
 Exopeptidase : caboxypeptidase A, carboxypeptidase B
 Endopeptidases : Trypsin, chymotrypsin

Exercise: Determination of peptide sequence

A peptide was cleaved into smaller fragments using chymotrypsin and CnBr. The fragments were separated by
HPLC and then sequenced by Edman degradation. Determine the sequence of the original peptide from the
following fragment sequences.

Cleavage with chymotrypsin Cleavage with CnBr

EAGPDGMECAF ECAFHR
GPF CKWEAGPDGM
EAAMCKW IAHTYGPFEAAM
HR
IAHTY

- Align the peptides

IAHTYGPEAAMCKW EAGPDGMECAFHR

REAGENT (SOURCE) SPECIFICITY

Trypsin (bovine pancrease) Lys, Arg (C)
Chymotrypsin (bovince pancrease) Phe, Trp, Tyr (C)
Staphylococcus V8 protease Glu, Asp (C)
Pepsin (porcine pancrease) Phe, Trp, Try (N)
Cyanogen bromide (chemical)(CnBr) Met (C)
X-Ray Diffraction

 a technique used to determine the property of the material such as structure.

ADDITIONAL NOTES

One of the biggest challenges in studying the detailed structure and properties of proteins is their
isolation and purification from cells and tissues with a highly complex composition and organization. Latest
refinements in the technology of protein purification and characterization have made this process easier and
quicker for researchers in this field. The extraction and isolation of proteins from cells and tissues is the first
important step towards the detailed study of proteins. Proteins are extracted from cells and tissues by
suspending them in the appropriate buffer and making a crude homogenate which can then be subjected to a
series of subcellular or differential centrifugation steps to remove cell debris and separate the various cell
fractions namely nuclear, mitochondrial, and microsomal fractions based on their sedimentation behavior when
subjected to increasing g forces (forces of gravity) in a centrifuge. The fraction which contains the protein of
interest is then chosen and the crude protein is salted out using ammonium sulfate, a common reagent used
to precipitate proteins from solution. The protein of interest will precipitate at specific percentage ammonium
sulfate saturation and can be localized in a particular fraction by following its activity. When localized, the
fraction is then dialyzed to remove excess ammonium sulfate, dried, and taken up in the appropriate buffer. It
is then ready for further purification and characterization steps which can involve a combination of high
performance liquid chromatographic and electrophoretic techniques. Once a pure sample of the protein is
obtained, it can now be subjected to other more sophisticated techniques such as mass spectrometry, protein
sequencing or X-ray diffraction techniques for further study of its structure and function
Proteins are extracted, isolated, and purified from cells or tissues by suspending the starting sample in
the appropriate buffer and homogenizing it in a Potter-Elvehjem tissue homogenizer or a similar device on ice
at 0-4oC until a crude homogenate is obtained. Thecrude homogenate is then subjected to subcellular or
differential centrifugation in a refrigerated centrifuge for separating proteins from a solution according to their
size, shape, density, viscosity of the medium and speed of the rotor or force of gravity. The various fractions
that can be obtained are the cell debris, nuclear fraction containing nuclei, mitochondrial fraction containing
mitochondria, lysosomes and peroxisomes, and microsomal fraction containing fragments of plasma
membrane, endoplasmic reticulum, and ribosomes.
Thus, the chosen fraction will be that fraction containing the protein of interest depending on its localization in
the cell. The chosen fraction contains many other molecules aside from protein, thus proteins are salted out or
precipitated using different percentage saturation of ammonium sulfate, a common reagent used to precipitate
proteins out from solution. As more ammonium sulfate is added, the proteins precipitate as ionic and polar
bonds are broken. Eventually one obtains different ammonium sulfate “cuts”, for example at 0-20%, 21-40%,
41-60%, 61-80%, 81-100% ammonium sulfate saturation and the protein is localized in a particular fraction by
following its activity. When localized, the fraction is then dialyzed to remove excess ammonium sulfate, dried,
and taken up in the appropriate buffer. This fraction contains the protein of interest as well as other proteins
with similar electrostatic characteristics and is ready for further purification by column chromatography and
electrophoresis.
Protein purification methods separate proteins based on charge, size, hydrophobicity, and specific
binding. Column chromatography may involve ion exchange (charge), gel filtration (size), reversed phase
(hydrophobicity), and affinity (specific binding). Chromatography may be done using high performance liquid
chromatography or fast performance liquid chromatography which involves separation of proteins by
partitioning them between the stationary phase and the mobile liquid phase. The proteins are detected by
UV/Visible absorption or fluorescence detector depending on the protein of interest. The detection system is
connected to a computer for integration and display of peaks. The protein of interest is followed by following
its activity or biological function in the fractions and the active fractions collected. Gel electrophoresis (native
polyacrylamide gel electrophoresis (PAGE), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-
PAGE) or isoelectrofocusing (IEF), and western blotting) is based on charge, size or both. It is used to visualize
protein peaks and assess the purity of the protein of interest. Once purity has been established, the protein is
then subjected to mass spectrometry, protein sequencing, or X-ray diffraction technique to describe its
primary, secondary, tertiary and quaternary structures.