Mary K. Campbell Shawn O.

Farrell
international.cengage.com/

Chapter Five Protein Purification and Characterization Techniques

Paul D. Adams University of Arkansas

Isolation of Proteins from Cells

Many different proteins exists within one cell

Many steps needed to extract protein of interest, and separate from many contaminants Before purification begins, protein must be released from cell by homogenization

How We Get Proteins Out of Cells

Salting Out
After Proteins solubilized, they can be purified based on solubility (usually dependent on overall charge, ionic strength, polarity Ammonium sulfate (NH4SO4) commonly used to salt out Takes away water by interacting with it, makes protein less soluble because hydrophobic interactions among proteins increases Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest (30, 40, 50, 75% increments) One fraction has protein of interest

Differential Centrifugation
Sample is spun, after lysis, to separate unbroken cells, nuclei, other organelles and particles not soluble in buffer used Different speeds of spin allow for particle separation

Column Chromatography
Basis of Chromatography
Different compounds distribute themselves to a varying extent between different phases

Interact/distribute themselves In different phases 2 phases: Stationary: samples interacts with this phase

Mobile: Flows over the stationary phase and carries along with it the sample to be separated

Column Chromatography

Size-Exclusion/Gel-Filtration
Separates molecules based on size. Stationary phase composed of cross-linked gel particles. 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 column before smaller ones.

Size Exclusion/Gel-filtration (Contd)

Affinity Chromatography
Uses specific binding properties of molecules/proteins Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to protein

Group Specific Affinity Resins

Ion Exchange
Interaction based on overall charge (less specific than affinity) Cation exchange Anion exchange

Electrophoresis
Electrophoresis- charged particles migrate in electric field toward opposite charge Proteins have different mobility:
Charge Size

Shape

Agarose used as matrix for nucleic acids Polyacrylamide used mostly for proteins

Electrophoresis (Contd)
Polyacrylamide has more resistance towards larger molecules than smaller Protein is treated with detergent (SDS) sodium dodecyl sulfate Smaller proteins move through faster (charge and shape usually similar)

Isoelectric Focusing
Isolectric focusing- based on differing isoelectric pts. (pI) of proteins

Gel is prepared with pH gradient that parallels electricfield. What does this do?
Charge on the protein changes as it migrates. When it gets to pI, has no charge and stops

Primary Structure Determination

How is 1 structure determined? 1) Determine which amino acids are present (amino acid analysis) 2) Determine the N- and C- termini of the sequence (a.a sequencing) 3) Determine the sequence of smaller peptide fragments (most proteins > 100 a.a) 4) Some type of cleavage into smaller units necessary

Primary Structure Determination

Protein Cleavage
Protein cleaved at specific sites by: 1) Enzymes- Trypsin, Chymotrypsin

2) Chemical reagents- Cyanogen bromide

Enzymes:
Trypsin- Cleaves @ C-terminal of (+) charged side chains Chymotrypsin- Cleaves @ C-terminal of aromatics

Peptide Digestion

Cleavage by CnBr
Cleaves @ C-terminal of INTERNAL methionines

Determining Protein Sequence

After cleavage, mixture of peptide fragments produced. Can be separated by HPLC or other chromatographic
techniques
Use different cleavage reagents to help in 1 determination

Peptide Sequencing
Can be accomplished by Edman Degradation

Relatively short sequences (30-40 amino acids) can be determined quickly

So efficient, today N-/C-terminal residues usually not done by enzymatic/chemical cleavage

Peptide Sequencing