Protein Analysis

Methods of protein purification and analysis

• Purification- cell fractination, chromatography, ultrafiltration

• Analysis- spectroscopy, SDS-PAGE, x-ray crystallography

Purpose of protein purification

• Study protein structure, function and interactions

• Essential in understanding disease mechanisms and drugs development

• Biopharmaceutical manufacturing - insulin, growth hormone

Study of proteins

• Control all major cellular functions, important in drug manufacturing

• Must be purified to determine structure and mechanism of action

• Common characteristics- amino acid structure, 3D shape

• Differ in size, shape, net charge, hydrophobicity, stability (resistance to heat), isoelectric point (pH at which they carry no charge)

• Aspirin inhibits COX to prevent prostaglandin formation (inflammation)

Initial considerations in protein purification

• Proteases - enzymes that digest proteins

• Source - bacteria, mammalian cells, biological fluids like blood, saliva, tears

• Can be isolated from original source (native) or expressed via genetic engineering (recombinant)

Cell lysis

• Detergent- physical

• Sonication- sound waves

• Osmotic shock- hypoosmotic solution

• Should leave membrane bound organelles intact, removed by centrifugation, releases proteins from cell

Differential centrifugation

• Centrifugation at increasing speeds to yield pellets of specific organalles

• Low speed - nuclei

• Medium - mitochondria

• High - lysosomes

• Supernatant - soluble target proteins, assay can be performed to identify where it is if not soluble

Column chromatography

• Stationary phase- solid, packed into a column

• Mobile phase- liquid or gas, carries material, flows through column

• Separation based on compounds interaction with stationary phase, large proteins come out first

• Partition chromatography- smaller molecules partition into pores e.g. gel filtration (molecular size)

• Adsorption chromatography- molecules adsorb onto stationary phase e.g. ion exchange (charge), affinity (ligand), hydrophobic interaction

SDS-PAGE

• Used to monitor protein purity

• Denatures protein, separates by mass

• Can resolve many proteins, individual bands can be excised and sequence

• Molecular weight determined by Rf calculation

Hydrodynamic radius

• Effective radius a protein has as it tumbles in solution, if proteins were strictly spherical it would correlate directly with their MW

• Asymetrical proteins may appear larger that spherical proteins of the same MW, elute earlier on a get filtration column

Gel filtration principles

• Filters based on size

• Vo - void volume (outside resin)

• Vi - volume inside resin

• Vt - total column volume (Vi + Vo) (Vg negligable)

• Ve - elution volume

• Kav - partition coefficient (Ve - Vo) / (Vt - Vo), larger proteins closer to 0, smaller closer to 1

• Large proteins elute first, too big to enter pores

Gel filtration procedure and considerations

• Equilibrate column with buffer that will be used in reaction, only one buffer with optimum pH and NaCl added to prevent non-specific electrostatic interactions

• Load concentrated (but less than 50mg/ml) sample onto column, less than 2% of Vt

• Wash continiously with buffer until proteins separate and elute

• Chromatogram will show order of elution

• Only about 10 proteins can be resolved

• Membrane proteins may not behave as expected due to detergents

Gel matrices used and applications of gel filtration

• De-salting - salts can be separated from proteins due to their small size

• Estimating molecular weight - load protein standards, plot MW vs elution time or volume and use graph to estimate

• Separating mixture of proteins by MW

• Gel matrices used - agarose (sepharose), dextran (sephadex), polyacrylamide (biogel)

Isoelectric point

• Proteins have surface charges based on amino acid composition (acidic vs basic) and environmental pH

• Isoelectric point - pH at which the net charge is zero

• If the pH is above the pl the protein has a negative charge

• If the pH is below the pl the protein has a positive charge

• Determined by algorithms that compare sum of acidic and basic side chains or isoelectric focusing, proteins migrate through pH gradient and stop where the net charge is 0

Principle of ion exchange chromatography

• Separates proteins based on electrical charge, can separate molecules that differ by just one charged amino acid

• The resin used is charges - cation exchanger is negatively charged and binds positive proteins, anion exchanger opposite

• pH of buffer is adjusted to ensure protein has the charge the resin requires e.g. pH<pl so protein is positively charged

• Elution - altering pH causing the protein to gain or lose charge, increasing NaCl concentration, ions will compete with protein

Myoglobin purification via ion exchange

• Add mince to buffer and strain, pl of 7

• Centrifuge and remove supernatant (myoglobin)

• Equilibrate column with pH 5.6 buffer, myoglobin becomes positively charged and binds to sephadex

• Weak cation exchanger

• Eluted as pH 7.5

• Lysozyme - pl 9.4, pH 8.2, elution at 10.2

What are ion exchange columns made of

• Backbone material - gives structure e.g. polystyrene, agarose

• Functional group (charged) - strong (stays charged over wide pH range) or weak anion or cation exchangers, does not indicate binding ability