BIOCHEMISTRY: PROTEIN PURIFICATION AND CHARACTERIZATION TECHNIQUES
EXTRACTING PURE PROTEINS FROM CELLS
(How do we get the proteins out of the cells?)
- Disruption of cells is the first step in protein purification. The various parts of cells can be separated by centrifugation. This is a useful step because proteins tend to
occur in given organelles. High salt concentrations precipitate groups of proteins, which are then further separated by chromatography and electrophoresis.
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PERCENT RECOVERY – a measurement of the amount of an enzyme recovered at each step of a purification experiment
HOMOGENIZATION – the process of breaking cells open to release the organelles
Different ways of homogenization of cells:
Blending with a suitable buffer
Potter-Elvejhem Homogenizer
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Sonication
Cycles of freezing and thawing
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Releases soluble proteins and various subcellular organelles
A thick walled test tube with a tight fitting plunger
The squeezing of the homogenate around the plunger breaks
open cells, but it leaves many of the organelles intact
Involves using sound waves to break open the cells
Rupture the cells
After Homogenization of cells
o DIFFERENTIAL CENTRIFUGATION - a process in which ruptured cells are centrifuged several times increasing the force of
gravity each time
o SALTING OUT – a purification technique for proteins based on differential solubility in salt solutions
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Ammonium sulfate is the most common reagent used to “salt out”
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Takes away water by interacting with proteins
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Makes protein less soluble because hydrophobic interactions increases among proteins
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Addition of salt – increases saturation
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Different set of proteins precipitates
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Centrifuge and save the set of proteins
COLUMN CHROMATOGRAPHY
(What are the different types of chromatography?)
- Gel-filtration chromatography separates proteins based on size. Ionexchange chromatography separates proteins based on net charge. Affinity chromatography
separates proteins based on their affinity for specific ligands. To purify a protein, many techniques are used and often several different chromatography steps are used
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Greek “chroma” (color), “graphien” (to write)
Based on the fact that different compounds can distribute themselves to varying extents between different phases, or separable
portions of matter
o Stationary Phase
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substance that selectively retards the flow of the sample, effecting the separation
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samples interacts with this phase
o Mobile Phase
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portion of the system in which the mixture to be separated moves
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flows over the stationary phase and carries with it the sample to be separated
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COLUMN CHROMATOGRAPHY
o a form of chromatography in which the stationary phase is packed in a column
o The sample is a small volume of concentrated solution that is applied to the top of the column; the mobile phase, called the
eluent, is passed through the column. The sample is diluted by the eluent, and the separation process also increases the
volume occupied by the sample.
DIFFERENT TYPES OF CHROMATOGRAPHY
o SIZE-EXCLUSION CHROMATOGRAPHY or GEL-FILTRATION CHROMATOGRAPHY
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Separates molecules based on size (molecular weight)
 Stationary phase composed of cross-linked gel particles (Beads)
 Two polymers :
→ Dextran or Agarose
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Dextran (Sephadex)
a complex polysaccharide that is often used in
column chromatography resins
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Agarose (Sepharose)
a complex polysaccharide used to make up resins
for use in electrophoresis and in column
chromatography
→ Polyacrylamide (Bio-Gel)
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a form of electrophoresis in which a polyacrylamide
gel serves as both a sieve and a supporting medium
 Extent of cross-linking can be controlled to determine pore size
 Smaller molecules enter the pores and are delayed in elution time
 Larger molecules do not enter and elute from the column before smaller ones
 ADVANTAGES
 Separate molecules based on size
 Estimate molecular weight by comparing sample with a set of
standards
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AFFINITY CHROMATOGRAPHY
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Uses specific binding properties of molecules/proteins
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Stationary phase has a polymer that is covalently linked to a compound called ligand
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Ligands bind to desired protein or vice versa
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Proteins that do not bind to ligand elute out
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The bound protein can then be eluted from the column by adding high concentrations of the ligand in soluble form, thus
competing for the binding of the protein with the stationary phase
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The protein binds to the ligand in the mobile phase and is recovered from the column
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This protein–ligand interaction can also be disrupted with a change in pH or ionic strength
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A convenient separation method and has the advantage of producing very pure proteins
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Very expensive
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ION-EXCHANGE CHROMATOGRAPHY
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A method for separating substances on the basis of charge
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the interaction is less specific and is based on net charge
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An ion-exchange resin has a ligand with a positive charge or a negative charge
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Cation exchanger a type of ion-exchange resin that has a net negative charge and binds to positively charged molecules
flowing through the column. Bound to Na+ or K+ ions.
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Anion exchanger a type of ion-exchange resin that has a net positive charge and binds to negatively charged molecules
flowing through it. Bound to Cl- ions.
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Column is equilibrated with buffer of suitable pH and ionic strength
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Exchange resin is bound to counterions
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Proteins – net charge opposite to that of exchanger stick to column
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No net charge or same charge elute -first
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HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
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A sophisticated chromatography technique that gives fast and clean purifications
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Exploits the same principles seen with other chromatographic techniques, but very high resolution columns that can be run
under high pressures are used
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Reverse phase HPLC a form of high performance liquid chromatography in which the stationary phase is nonpolar and the
mobile phase is a polar liquid
ELECTROPHORESIS
(What is the difference between agarose gels and polyacrylamide gels?)
- Agarose gel electrophoresis is mainly used for separating nucleic acids, although it can also be used for native gel separation of proteins. Acrylamide is the usual medium
for protein separation. When acrylamide gels are run with the chemical SDS, then the proteins separate based on size alone.
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Electrophoresis
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Charged particles migrate in electric field toward opposite charge
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a method for separating molecules on the basis of the ratio of charge to size
Agarose - matrix for nucleic acids
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Charge, size, shape
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Agarose matrix has more resistance towards larger molecules than smaller
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Small DNA move faster than large DNA
Polyacrylamide - proteins
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Charge, size, shape
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Treated with detergent (SDS) sodium dodecyl sulfate – gains –ve charge
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Random coil – shape
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Polyacrylamide has more resistance towards larger molecules than smaller
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Small proteins move faster than large proteins
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SDS–polyacrylamide-gel electrophoresis (SDS–PAGE) an electrophoretic technique that separates proteins on the basis of size
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native gel one without SDS or another compound that would denature the proteins being separated
Isolectric focusing
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Gel is prepared with pH gradient that parallels electric-field
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Charge on the protein changes as it migrates across pH
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When it gets to pI, has no charge and stops
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Separated and identified on differing isoelectric pts. (pI)
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A combination known as two-dimensional gel electrophoresis (2-D gels) allows for enhanced separation by using
isoelectric focusing in one dimension and SDS–PAGE run at 90° to the first (Figure 5.13).
DETERMINING THE PRIMARY STRUCTURE OF A PROTEIN
(Why are the proteins cleaved into small fragments for protein sequencing?)
- The Edman degradation has practical limits to how many amino acids can be cleaved from a protein and analyzed before the resulting data become confusing. To avoid
this problem, the proteins are cut into small fragments using enzymes and chemicals, and these fragments are sequenced by the Edman degradation.
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Step how is 1˚ structure determined?
1. Determine which amino acids are present and in what proportions (amino acid analyzer)
2. Specific reagents - determine the N- and C- termini of the sequence
3. Cleave - determine the sequence of smaller peptide fragments (most proteins > 100 amino acids)
4. Some type of cleavage into smaller units necessary
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What is Edman degradation?
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a method for determining the amino acid sequence of peptides and proteins
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Cleaving of each amino acid in sequence followed by their subsequent identification and removal
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Becomes difficult with increase in number of amino acids
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Amino acid sequencing - Cleave long chains into smaller fragments
Cleavage of the Protein into Peptides
o Trypsin a proteolytic enzyme specific for basic amino acid residues as the site of hydrolysis. Cleaves @ C-terminal of (+)
charged side chains/R-groups
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Chymotrypsin a proteolytic enzyme that preferentially hydrolyzes amide bonds adjacent to aromatic amino acid residues.
Cleaves @ C-terminal of aromatics
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Cyanogen bromide a reagent that cleaves proteins at internal methionine residues. Cleaves @ C-terminal of INTERNAL
methionines. Sulfur of methionine reacts with carbon of cyanogen bromide to produce a homoserine lactone at C-terminal
end of fragment.
Use different cleavage reagents to help in 1˚ determination
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Determination of primary structure of protein
o After cleavage, mixture of peptide fragments are produced.
o Sequences can overlap – peptides can be arranged in proper order after all sequences have been determined
o Can be separated by HPLC or other chromatographic techniques
Peptide sequencing by Edman Degradation
o Can be accomplished by Edman Degradation
o Sequencer - relatively short sequences (30-40 amino acids in 30-40 picomoles) can be determined quickly
o N-/C-terminal residues - not done by enzymatic/chemical cleavage
o Edman’s reagent – Phenyl isothicocyanate
o Reacts with peptide’s N-terminal residue – cleaved
o Leaves rest of peptide intact – phenylthiohydantoin derivative of amino acid
o Same treatment to 2, 3,4….40
o Automated Sequencer – Process repeated
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Sequencer an automated instrument used in determining the amino acid sequence of a peptide or the nucleotide
sequence of a nucleic acid
PROTEIN IDENTIFICATION TECHNIQUES
(What are some common protein identification techniques?)
- There are several ways to identify proteins. Mass spectrometry measures the charge to mass ratios of atoms in molecules and can identify a protein down to its atomic
level. Enzymelinked immunosorbent assay (ELISA) and western blots both exploit the specific binding of antibodies to a protein with subsequent visualization of the
antibody-protein complex. In ELISA this is carried out in a microtiter plate. With western blots, proteins are first separated with gel electrophoresis and then the bands
transferred to a membrane, such as nitrocellulose. A very powerful technique uses thousands of proteins stuck on a slide, called a protein microarray or protein chip.
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ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
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Is based on reactions between proteins and antibodies
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Basic idea is that vertebrates produce proteins, called antibodies, when they encounter foreign molecules, including other
proteins.
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The antibodies bind very specifically to the proteins that elicited their creation
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WESTERN BLOT
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Refers to the transfer of proteins from an electrophoresis gel, usually SDS-PAGE, onto a thin membrane of nitrocellulose or some
other absorbent material
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Western blot, comes from a “tongue in cheek” derivation of all blotting techniques
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Was first named as southern blot
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PROTEIN CHIPS
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Protein chips also called protein microarrays; small plates of a few centimeters on a
side that can have tens of thousands of proteins implanted
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May have 30,000 separate samples stuck on a chip a few centimeters on a side.
Fluorescence is the most common way to see the results, as shown in figure 5.25.
PROTEOMICS
(How do the individual protein techniques combine to study proteomics?)
- Proteomics refers to our attempts to study the entire complement of proteins being produced by a cell at a certain
time under specified conditions. All of the techniques studied in this chapter are involved in fishing out the identity and
nature of the many thousands of proteins in a cell.
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Proteomics study of interactions among all the proteins of the cell
Proteome the total protein content of the cell
Subdivided into three basic types
o Structural proteomics
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Offers a detailed analysis of the structure of the proteins being produced
o Expression proteomics
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Analyzes the expression of proteins, and frequently considers their expression under different cellular conditions
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A major contributor to our understanding of metabolism and disease
o Expression proteomics
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Analyzes the expression of proteins, and frequently considers their expression under different cellular conditions
Process on how do the individual protein techniques combine to study proteomics:
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They created proteins they called “the bait,” shown as protein 1 in figure 5.26.
These were tagged with an affinity label and allowed to react with the other cell components.
The tagged bait proteins were then allowed to bind to an affinity column.
In binding to the column, they took any other bound proteins with them.
The bound complex was eluted from the column, then purified with SDS–page.
The bands were excised and digested with trypsin.
After digestion, the pieces were identified with mass spectrometry.
The identities of the proteins associated with the bait protein were established.