Protein Purification and Determination

Protein Purification and
Determination of Protein
Structure
Lesson Learning Outcomes
Upon completion of this lecture, students
should be able to:
• Know the procedure of protein extractions
• Differentiate types of chromatography
• Know the electrophoresis
Protein Purification
• Differential centrifugation
• Used for separation of cell components using
different speed of centrifugation
– Different centrifugational force will separate larger
cell components from smaller ones
– Low speed to separate large cell components
– High speed to separate smaller cell components
Homogenization
• Before the real purification steps can begin, the
protein must be releases from the cells and
subcellular organelles through differential
centrifugation
• In general, separation techniques focus on size,
charge and polarity.
• Proteins will be purified either through size-exclusion
chromatography, affinity chromatography, isoelectric
points, High Performance Liquid Chromatography
(HPLC).
Chromatography
• Greek chroma = color
• Greek graphein = to write
• Early technique used to separate plant
pigments with easily visible colors
• It is based on the fact that the different
compounds can distribute themselves to
different phases
• Stationary phase & mobile phase
Column chromatography
Size-exclusion chromatography
• Also called gel-filtration chromatography
• Separate molecules on the basis of size
• Sort proteins of varied molecular weights
• The stationary phase consists of cross-linked gel
particles
– Consist of two kinds of polymers
• Carbohydrate polymer – dextran (Sephadex) or
agarose (Sepharose)
• Polyacrilamide (BioGel)
• Extend of cross-linking can be controlled to
determine the pore size needed.
Affinity chromatography
• Uses the specific binding properties of many
proteins.
• Polymeric material used as the stationary phase.
• Polymer is covalently linked to ligand that binds
specifically to the desired protein.
• Other proteins can be easily eluted out with
buffer.
• Desired proteins will then be eluted with high
concentration of ligands in soluble form.
• Producing very pure protein.
Ion-exchange chromatography
• The interaction is less specific and is based on net

charge
• An ion-exchange resin will have a ligand with a
positive or a negative charge
– A negatively charged resin = cation exchanger
• Bound to Na+ or K+
– A positively charged resin = anion exchanger
• Bound to Cl-
Electrophoresis
• Is based on the motion of charged particles in

an electric field toward an electrode of
opposite charge
• Most common support is a polymer of agarose
or acrylamide
• Agarose- nucleic acids / small protein
• Acrylamide- protein
Isoelectric focusing
• Is another variation of gel electrophoresis

• Different proteins have different isoelectric
points (pH at which a particular molecule
carries no net electrical charge)
– At the pH, the number of +ve charges exactly
balances the number of –ve charges
• In isoelectric focusing, a gel is prepared with
a pH gradient that parallels the electric field
gradient
2D Gel electrophoresis
Determine the primary structure of a
protein
 Step 1: Determine which amino acid is present and in
what proportion
 Heat protein in acid (6M HCl) at 100-110 oC for 12-
36 hrs to hydrolyze peptide bonds
 Use amino acid analyzer
• Will give the quantitative and qualitative
analysis of amino acids
• Will separate mixture of amino acids either by
ion-exchange chromatography or HPLC (high
performance liquid chromatography)
 Step 2: identification of the first amino acid –
Sanger degradation
 Treat protein with thiol – breaks disulfide
bond  polypeptide
 Add 2,4-dinitro-fluorobenzene – binds to the
first amino acid of the polypeptide
 Hydrolysis of the polypeptide  individual
amino acids
 Identify the amino acid with 2,4-dinitro-
fluorobenzene as the first amino acid
 Step 3 and 4 : cleave proteins into smaller
fragments  Edman degradation
 Enzyme trypsin cleave peptide bonds
preferably at amino acids that have positively
charged R groups (eg: lysine and arginine),
where the charged side chain ends up on the
C-terminal side
 Chymotrypsin cleaves peptide bonds of
aromatic amino acid where the aromatic
amino acid end at the c-terminal
 Automated sequencing of protein fragments
INTERACTIONS
Forces that stabilize the tertiary structure of proteins.

Note that the helical structure and sheet structure are two kinds of backbone
hydrogen bonding. Although backbone hydrogen bonding is part of secondary
structure, the conformation of the backbone constrains the possible arrangement of
the side chains.
Determination the tertiary structure of a
protein
 X-ray crystallography – measure the three-
dimensional (3-D) density distribution of electrons in
the protein, in the crystallized state.
 2D Nuclear magnetic resonance (2D NMR)-ability to
distinguish between the overlapping signals that
exist in larger molecules.
 NMR spectroscopy can quantitatively analyze
mixtures containing known compounds. For
unknown compounds, NMR can either be used to
match against spectral libraries or to infer the basic
structure directly.
End of lecture
Please Read up page 117-135

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Protein Purification and

• The interaction is less specific and is based on net

• Is based on the motion of charged particles in

• Is another variation of gel electrophoresis

Forces that stabilize the tertiary structure of proteins.

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