Module 04: Protein Purification & Sequencing

Why is protein purification important?

To study protein structure, function, mutations, and for drug development

What two main properties are proteins separated by?

Size and charge

How do you estimate molecular weight of a protein?

MW = (# residues) x 110 Daltons

What are the 5 main steps in protein purification?

1) isolate protein, 2) detect protein, 3) assay activity, 4) separation, 5) quantitation

How are proteins isolated?

Cell lysis (mechanical or chemical and solubilization, under proper pH, temp, and with protease inhibitors)

What amino acids absorb UV light?

Phe, Tyr, Trp (260-280 nm)

How does the Bradford Assay work?

Coomassie blue binds proteins, changing absorbance from 465 to 595 nm (high sensitivity)

What is the Beer-Lambert law?

A = ElC; relates absorbance to concentration

What is ‘salting out’?

Precipitating proteins at different salt concentrations based on solubility

How does ion exchange chromatography work?

Proteins binds columns with opposite charge; eluted by salt or pH change

What’s the difference between anion and cation exchange?

Anion = binds — charged proteins; cation = binds + charge proteins

What is hydrophobic interaction chromatography?

Uses high salt to promote nonpolar protein interaction with phenyl columns; eluted by decreasing salt

How does gel filtration (size exclusion) work?

Large protein elute first; small one travel through porous beads and elute later.

What is affinity chromatography?

Uses ligands bounds to resin to binds specific proteins; eluted by competition

How is purification % calculated?

(Amount of target protein/total protein) x 100

What is specific activity?

Enzyme activity per mg of protein; used to track purification

What does SDS do?

Denatures proteins and gives them uniform negative charge

How are proteins separated in SDS-PAGE?

By size only; small protein travel farthest

What’s the purpose of a molecular weight ladder?

To estimate unknown protein size by comparison

How does isoelectric focusing work?

Proteins migrate in a pH gradients until they reach their pI (net charge= 0)

What does 2D gel electrophoresis combine?

Isoelectric focusing (1D) + SDS-PAGE (2D); separates proteins by pI and size

Why sequence proteins?

To determine structure, function, evolutionary relationships, or detect mutations

What do proteases do?

Cleave peptides bonds

What’s the difference between endo- and exopeptidases?

Endo = cleave internal bonds; exo = cleave N- or C-terminal residues

What residues does trypsin cleave after?

Arginine ® and lysine (K)

What residues does chymotrypsin cleave after?

Phe, Trp, Tyr

What does CNBr cleave after?

Methionine (Met)

What are zymogens?

Inactive precursors of proteolytic enzymes

Why are zymogens necessary?

To prevent unintended tissue digestion before reaching digestive organs

What does Edman degradation do?

Sequentially removes and identifies the N-terminal residue of a peptide

What reagent is used in Edman degradation?

Phenylisothiocyanate (Edman’s reagent)

What chemical breaks disulfide bonds?

Beta-mercaptoethanol

What prevents disulfide reformation?

Iodoacetate

What tool is used to analyze amino acid composition?

High Performance Liquid Chromatography (HPLC)

What determine N- and C-terminal residues?

N-term: reacts with Dansyl Chloride or FDNB; C-term identified using carboxypeptidases A and B

How does MS help sequence peptides?

Measures m/z (mass-to-charge) ratios to identify residues

What is ESI-MS?

Electrospray ionization mass spectrometry — ionizes proteins in solution