Exam next week: Chapter 4? Lab next week Today: protein purification/general strategies

Protein purification: Separate relevant proteins from all other cellular proteins

Chromatographic separation of proteins Exploit differences in physical/chemical characteristics Charge Size (effective size) Ligand binding (ability to form strong interactions with other molecules) Hydrophobicity

Chromatography Typically: add a mixture in a mobile phase to some sort of stationary phase Some components have affinity for the mobile phase, some for the stationary phase Mobile and stationary phase separate

Protein mixture (A,B,C, other) in mobile phase Proteins have different affinities for stationary/mobile phases Proteins elute at different times spectrophotometer

Chromatogram: Two pure proteins 3-7 minutes 10-15 minutes A280 Retention time

Types of protein chromatography Ion-exchange Cation Anion Size-exclusion a.k.a. Gel filtration Affinity

Affinity chromatography Stationary phase: ligand tightly bound to an insoluble ‘resin’ Protein of interest has an affinity for the ligand ‘Natural’ affinity: eg. an ATP-binding protein ‘Artificial’ affinity: eg. use genetic manipulation to add a binding site

Ion exchange chromatography Mobile phase: aqueous Important variables: Salt concentration pH Stationary phase: Insoluble “bead” with covalently-bound, charged group

Examples of ion exchange resins + Cl- Quaternary amine Insoluble polymer Ion exchanger Counter-ion O - S O Na+ Sulfonate O

Ion exchange Cation exchange Anion exchange Matrix is negatively charged Counter-ions are cations Selective binding of positively-charged proteins Anion exchange Matrix is positively charged Counter-ions are anions Selective binding of negatively-charged proteins

Anion exchange - + Counter-ions: anions (neg. charge) + + Cl- Cl- + Proteins with net negative charge have affinity for stationary phase “Exchange” of anions on resin Proteins with net positive charge have relatively higher affinity for mobile phase + Cl- Cl- Cl- + + +

Cation exchange The higher the positive charge, the longer a protein takes to elute

Changing of the mobile phase Low to high salt concentration Protein of interest has net positive charge Strong adherence to stationary phase (cation exchange) “Stuck” on the column Increase salt concentration: decrease protein’s affinity for stationary phase Move protein to mobile phase & recover it

Anion Exchange Chromatography Anionic (negatively charged) proteins adhere to a positively charged resin ‘Exchange’ occurs when another source of anions (eg. salt) is introduced Proteins with different charge characteristics elute (become ‘unstuck’) at different salt concentrations ‘Separation’ of proteins is achieved because different proteins (1) don’t stick at all, (2) stick weakly, or (3) stick strongly depending on their biophysical characteristics

Gel filtration/Size exclusion: Separation based on “size” Very large proteins (or protein complexes or other molecules) traverse the column quickly [elute first] Very small proteins have the largest accessible volume and the farthest to travel: traverse the column very slowly [elute last] Proteins/complexes between these extremes traverse the column at varying speeds

Gel Filtration/Size Exclusion Chromatography resin (stat. phase) Polymer beads with tiny ‘pores’

Gel Filtration/Size Exclusion

GF: Separation based on size