1. Protein purification: the basics Arvind Varsani

2. Reasons for protein purification To identify the FUNCTION of a protein To identify the STRUCTURE of a protein To use the use the purified product – INTERMIDIATE- in downstream reactions / processing To produce a COMMERCIAL product

3. Selection of protein source Starting material can be from –Animal tissue –Plant material –Biological fluids (e.g. blood, milk, sera) RECOMBINANT expression –Fermentation cultures (yeast, fungi, bacteria) –Cell cultures (animal cells, plant cells, insect cells)

4. Important Protein in low concentration in natural sources –Need to induce expression Or express recombinantly in various expression systems

5. Classification of proteins by structural characterisation Structural characteristicsExamplesComments MonomericLysozyme, growth hormoneUsually extracellular; often have disulphide bonds Oligomeric with identical subunitsGlyceralsehyde-3-phosphate dehydrogenas, catalase, hexokinase Mostly intracellular enzymes, rarely have disulphide bonds Oligomeric with mixed subunitsPetussis toxinAllosteric enzymes, different subunits have different functions Membrane bound – peripheralMitrochondrial ATPase, alkaline phosphatise Readily solubilised in detergents Membrane bound – intergralPorins, insulin receptorsRequires lipid for stability Membrane bound – conjugatedGlycoproteins, lipoproteins, nucleoproteins Many extracellular proteins contain carbohydrate

6. Classification of proteins by function FunctionExamples Amino acid storageSeed proteins (e.g. gluten), milk proteins (e.g. caesin) Structural – inertCollagen, keratin Structural – with activityActin, myosin, tubulin Binding – solubleAlbumin, hemoglobin, hormones Binding – insolubleSurface receptors (e.g. insulin receptor), antigens (e.g. viral coat proteins) Binding – with activityEnzymes, membrane transporters (e.g. ion pumps Relative abundance

7. Protein properties and their effect on development of purification strategies Sample and target protein propertiesInfluence on purification strategy Temperature stabilityNeed to work rapidly at lower temperatures pH stabilitySelection of buffers for extraction and purification (conditions for ion exchange, affinity or reverse phase chromatography) Organic solventsSelection of condition for reverse phase chromatography Detergent requirementsConsider effects on chromatographic steps and the need for detergent removal. consider choice of detergent Salt (ionic strength)Selection of conditions for precipitation techniques and hydrophobic interaction chromatography Co-factors for stability or activitySelection of additives, pH, salts and buffers Protease sensitivityNeed for fast removal of proteases or addition of inhibitors Sensitivity to metal ionsNeed to add EDTA or EGTA in buffers Redox sensitivityNeed to add reducing agents Molecular weightSelection of gel filtration media Charge propertiesSelection of ion exchange conditions Biospecific affinitySelection of ligand for affinity medium Post translational modificationsSelection of group specific affinity medium HydrophobicitySelection of medium for hydrophobic interaction chromatography

8. Yields for multi-step protein purifications 100 Limit the number of steps Optimise each step Be careful of the yield if the proceduce requires several steps

9. Key steps in purification Release of target protein from starting material Removal of solids to leave the protein in the supernatant Concentration of the protein Removal of contaminants to achieve the desired purity Stabilization of the target protein

10. Three phase purification strategy The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by 3 major purifications step. The number of steps will depend on the purification strategy, purity requirements and intended use of the protein

11. Protein analysis Tracking protein of interest and determining the yield during purification –Intended use of protein / source of starting material Physical studies e.g. x-ray, NMR, EM End product – pharmaceuticals

12. Analysis of protein purity Total protein Specific quantification –Activity assays –Binding assays Detection of impurities –HPLC –Gel electrophoresis Protein mass spectrometry

13. Methods for quantification of proteins in solution Assay methodUseful rangeComments NanoOrange assay100ng/ml to 10ug/ml  Samples can be read up to six hours later without any loss in the sensitivity  Low protein to protein signal variability  Detection not influenced by reducing agents or nucleic acid BCA method (Cu reduction) 0.5ug/ml to 1.5mg.ml  Samples must be read within 10 mins  Not compatible with reducing agents Bradford assay (dye binding) 1ug/ml to 1.5 mg/ml  Protein precipitates over time  High protein to protein signal variability  Not compatible with detergents Lowry assay1ug/ml to 1.5mg.ml  Lengthy, multi-step procedure  Not compatible with detergents, carbohydrates or reducing agents Absorbance at 280nm50ng/ml to 2mg/ml  High protein to protein signal variability  Detection influenced by nucleic acids and other UV absorbing contaminants

14. BSA assay (Bicinchoninic acid) The first step is a Biuret reaction which reduces Cu+2 to Cu+1 In the second step BCA forms a complex with Cu+1 which it purple colored and is detectable at 562 nm Bradford assay (coomassie dye binding) Absorbance shift in Coomassie Brilliant Blue G-250 (CBBG) when bound to arginine and aromatic residues The anionic (bound form) has absorbance maximum at 595 nm whereas the cationic form (unbound form) has and absorbance maximum at 470 nm

15. Lowry assay (Cu reduction)  Monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or if the wavelength is lowered, the absorbance of the peptide bond. Higher order structure in the proteins will influence the absorption The first step is a Biuret reaction which reduces Cu+2 to Cu+1 The second reaction uses Cu+1 to reduce the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). This is detectable in the range of 500 to 750 nm Absorbance at 280nm

16. Enzyme activity assays Continuous (kinetic assays) –No separation step

17. ELISA SDS

18. Cell disruption / breakage for protein release Extraction techniques are selected based on the source of protein (e.g. bacteria, plant, mammalian, intracellular or extra cellular) Use procedures that are as gentle as possible. Cell disruption leads to the release of proteolytic enzymes and general acidification Selection of an extraction technique often depends on the equipment availability and the scale of operation Extractions should be performed quickly, at sub-ambient temperatures in a suitable buffer to maintain pH and ionic strength Samples should be clear and free of particles before beginning chromatography

19. Cell disruption: source variations Tissues – variable Mammalian cells – easy Plant cells – some problems Microbial cells – vary, common Yeast and fungal cells – more difficult

20. Cell disruption: methods Chemical / enzymatic Cell lysis (osmotic shock and freeze thaw) Enzymatic digestion Blood cells Mammalian cells Fractional precipitation Extra cellular proteins Mechanical Hand and blade homogenizers tissue Sonicator / disruptors Grinding with abrasive plant/yeast Bad beaters / mill French press micro fluidizer

21. Lytic enzymes and detergents Lysozyme: disrupts bacterial cell walls (hydrolysis of peptidoglycans) leading to cell rupture –Effective with gram positive bacteria, gram negative generally require pre-treatment with a chelating agent such as EDTA Detergents: anionic and non-ionic detergents have been used to permeabilize gram negative cells. Detergents are required for the release of integral membrane proteins.

22. Simple shear methods Glass homogenizer (dounce, ten-broeck)

23. sonicator French pressure cell

24. Sample clarification Centrifugal sedimentation Coagulation and flocculation Filtration

25. Sedimentation Operates on the basis of density difference between components in a mixture (e.g. solids and liquid) Rate of sedimentation is dependent on: –Magnitude of different in component densities –Particle size, shape and concentration –Magnitude of centrifugal force –Flocculating of coagulating cells or organelles

26. Coagulation and flocculation Coagulation –Increase in particle size from the joining of like particles –Promote by reducing charge repulsion Addition of multivalent ions (e.g. Al 3+ ) Adjust pH to isoelectric point Flocculation –increase in particle size by addition of agents acting as bridges between particles –Generally polyelectrolytes that neutralise surface charges on particles and then link particles to form aggregates

27. Concentration of extracts Freeze drying Dialysis PEG precipitation Concentration / fractionation by salting out –Ammonium sulphate precipitation Ultraflitration –Desalting –Size fractionation

28. Protein purification Affinity chromatography –Binding to immobilised ligands e.g antibodies, co- factors Ion exchange chromatography –Anion (-) and cation (+) exchanger Hydrophobic interaction chromatography –Colum coated with hydrophobic fatty acid chains Size exclusion chromatography –Gel filtration Electrophoresis –SDS