BICH 605

1. BICH 605 Protein Purification • Sample Preparation • Extraction (grinding, detergent lysis, sonication) • Salt exchange (gel filtration, filters, dialysis) • Capture • Ion Exchange • Affinity • Hydrophobic Interaction • Intermediate Purification • Ion exchange • Hydrophobic Interaction • Polishing • Gel Filtration • Reversed phase

2. More Protein Protein Measurement: The Bradford Assay (Coomassie Blue Binding)

3. Protein Measurement: The Bradford Assay (Coomassie Blue Binding) Build a Standard Curve Protein Variation is Great

4. Protein Measurement: The Bicinchoninic Acid (BCA) Assay

5. CAPTURE METHODS • ION EXCHANGE • HYDROPHOBIC INTERACTION • AFFINITY CAPTURE

6. pI = ISOLECTRIC POINT BELOW pI NET CHARGE IS POSITIVE (a cation) ABOVE pI NET CHARGE IS NEGATIVE (an anion) ION EXCHANGE CHROMATOGRAPHY • ION EXCHANGE BINDINGIS BASED ON SURFACE CHARGES OF PROTEINS • PROTEINS ARE IONS • ELUTION IS CAUSED BY COMPETITION WITH COUNTER IONS IN BUFFER (OFTEN SODIUM CHLORIDE) • CHARGE IS A FUNCTION OF pH

7. ION EXCHANGE CHROMATOGRAPHY An example of Ion Exchange “Exchange” protein cation with a Cationic Counterion Protein acts like a CATION Protein acts like an ANION

8. ION EXCHANGE CHROMATOGRAPHY • IEX CAN BE EITHER ANION EXCHANGE OR CATION EXCHANGE • MANY DIFFERENT MATRICES AVAILABLE COMMERCIALLY • CHOOSE BASED ON STABILITY OF PROTEIN AT pH OF OPERATION AND PROPERTIES OF ‘OTHER’ PROTEINS IN MIXTURE • STRONG AND WEAK BINDERS CHARACTERIZE BINDING STRENGTH

9. ION EXCHANGE CHROMATOGRAPHY SET UP OF APPARATUS • COMPONENTS: • BUFFER RESERVOIRS • COLUMN FILLED WITH MATRIX • MONITOR & RECORDER (not shown) • FRACTION COLLECTOR (not shown)

10. ION EXCHANGE CHROMATOGRAPHY GRADIENT ELUTION TECHNIQUES HIGH SALT LOW SALT Linear Gradient Step Gradient Detector Fraction Collector animation

11. ION EXCHANGE CHROMATOGRAPHY FLOWRATE AND GRADIENT SLOPE EFFECT RESOLUTION Shallow Gradient; 8 ml/hr Generally achieve 5-10 fold enrichment of target proteins depending upon sample complexity and steepness of gradient. Often used as a first chromatographic step in purification as it can removed oppositely charged molecules immediately. Useful in presence of non-ionic detergents, urea and other chaotropes. Scaling options are wide. Fractions can be applied directly to hydrophobic interaction chromatography matrices (high salt). A “concentrating” method. Steep Gradient; 8 ml/hr Shallow Gradient; 20 ml/hr

12. CAPTURE METHODS • ION EXCHANGE • HYDROPHOBIC INTERACTION • AFFINITY CAPTURE

13. HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) HIC SEPARATES BIOMOLECULES BASED ON THE HYDROPHOBIC GROUPS ON THEIR SURFACES. Binding of biomolecules to the mildly hydrophobic surface of a HIC column is induced by the addition of high salt concentrations to the sample and equilibration buffer. Elution is effected by decreasing the salt concentration.

14. HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) ELUTION FROM HIC COLUMN IS BY DECREASING IONIC STRENGTH OF BUFFER (Note: This is OPPOSITE of Ion Exchange Chromatography). DIFFERENT SALTS MAY BE USED; AMMONIUM SULFATE IS MOST COMMONLY USED

15. HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) DIFFERENT FUNCTIONAL GROUPS ARE AVAILABLE WITH DIFFERENT DEGREES OF HYDROPHOBICITY

16. HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) DIFFERENT FUNCTIONAL GROUPS HAVE DIFFERENT SELECTIVITY Cannot be used with detergent containing buffers (hydrophobic column binds detergents to exclusion of proteins). Useful as a preceding step to IEX as it is eluted in low salt buffer. A “concentrating” method.

17. CAPTURE METHODS • ION EXCHANGE • HYDROPHOBIC INTERACTION • AFFINITY CAPTURE

18. AFFINITY CHROMATOGRAPHY A TYPE OF ADSORPTION CHROMATOGRAPHY IN WHICH THE MOLECULE TO BE PURIFIED IS SPECIFICALLY AND REVERSIBLY ADSORBED BY A COMPLEMENTARY BINDING SUBSTANCE (LIGAND) IMMOBILIZED ON AN INSOLUBLE SUPPORT (MATRIX). PURIFICATION IS OFTEN OF THE ORDER OF SEVERAL THOUSAND FOLD; RECOVERY OF ACTIVE MATERIAL CAN BE VERY HIGH.

19. AFFINITY CHROMATOGRAPHY HOW TO DESIGN AN AFFINITY MATRIX ANY COMPONENT CAN BE USED AS A LIGAND. IT IS IMPORTANT THAT THE IMMOBILIZED LIGAND RETAINS ITS SPECIFIC BINDING ACTIVITY. CORRECT CHOICE OF COUPLING GEL IS DICTATED BY BOTH THE TYPE OF GROUPS AVAILABLE ON THE LIGAND MOLECULE FOR COUPLING AND THE NATURE OF THE BINDING REACTION. IMMOBILIZATION SHOULD BE ATTEMPTED THROUGH THE LEAST CRITICAL REGION OF THE LIGAND. SPACER ARMS?

20. AFFINITY CHROMATOGRAPHY COUPLING REACTIONS WITH ACTIVATED RESINS • AFFIGEL RESINS (BIORAD) • Aqueous buffer, pH 3-10 • N-hydroxysuccinimide ester coupling reaction for ligands with primary amines • CYANOGEN BROMIDE-ACTIVATED RESIN (NO SPACER) • Aqueous buffer, pH 8-10 (more acidic is slightly less efficient, but slower spontaneous hydrolysis of ester) for ligands with primary amines

21. IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC) Immobilized metal affinity chromatography (IMAC), proteins and peptides separates on the basis of their affinity for metal ions which have been immobilized by chelation. Histidine and cysteine form complexes with the chelated metals around neutral pH (pH 6-8). Transitional metals are most often used for IMAC.

22. IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC) Binding is pH dependent and elution is carried out by reducing the pH or increasing ionic strength of the buffer. Another widely used elution method is to use EDTA in the buffer. This method is very useful and widely used to purify recombinant proteins that have been made as over-expressed, His-tag fusion proteins.

23. IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC) NICKEL IMAC HIS-TAG PURIFICATION SDS PAGE

24. POLISHING METHODS • GEL FILTRATION • REVERSED PHASE HPLC

25. Gel Filtration Chromatrography (GFC)(Size Exclusion Chromatrography) Principle • Gel filtration is performed using porous beads as the chromatographic support. GFC separates proteins based on size. • Separation is accomplished by the differential in ‘residence time’ a protein spends inside of or outside of the porous beads.

26. Gel Filtration Chromatrography The elution volume (Ve) of a protein can be calculated as a partition coefficient (Kav) using the following equation where Vt equals the sum of the external volume and the internal volume: A semi-logarithmic plot of the dependence of the partition coefficient (Kav) on molecular weight is illustrated here. The separation of proteins based on molecular weight will be greatest in the central linear region of this sigmoidal relationship, spanning Kav values between 0.2 and 0.8. This span is described as the fractionation range of a size-exclusion matrix.

27. Gel Filtration Chromatrography GEL FILTRATION MATRICES ARE AVAILABLE TO SEPARATE A WIDE RANGE OF MOLECULAR SIZES AND CONSTRUCTED FROM A VARIETY OF MATERIALS SEPHADEX™ Cross-linked dextrans (range 1-600 kDa) SUPERDEX™Dextran/agarose matrix (range 3-600 kDa) SEPHAROSE™ (range 10-40,000 kDa)

28. Gel Filtration Chromatrography GENERALIZED GFC SETUP animation

29. Gel Filtration Chromatrography GFC IS USEFUL FOR DESALTING PROTEIN SOLUTIONS (vs. DIALYSIS)

30. Gel Filtration Chromatrography EFFECT OF FLOWRATE AND BED HEIGHT ON RESOLUTION AND GEL FILTRATION CHROMATOGRAPHY • 1 vs 2 EFFECT OF FLOWRATE  • 2 vs 3 EFFECT OF BED HEIGHT 1 Generally, fewer than 10 proteins can be resolved from one another in the effluent of a size exclusion column. This being the case, it is wise to perform this method relatively late in a purification procedure when the numbers of proteins are relatively small and when the preceding step has fractionated the protein mixture on the basis of a completely different property. 2 3

31. END OF DAY 2