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Biochemistry 412 2004 20 February Lecture Analytical & Preparative Protein Chemistry II Proteins are Amphiphilic Macro-Ions Positively-charged basic residues (K, R, & H) Hydrophobic “patch” Macromolecular dimensions: ca. 40 Å Ligand binding pocket (active site) Negatively-charged acidic residues (E & D) >>> The charged groups, hydrophobic regions, size, and solvation affect the biophysical properties of the protein and largely determine its purification behavior. Chromatography Sample containing proteins or peptides Liquid flow Liquid flow Separation according to: -molecular weight/ size -charge -hydrophobicity -affinity Time 4:37 990909 1 2 3 4 5 5 Three Phase Strategy: An aid in developing the purification scheme Achieve final purity. Remove trace impurities, structural variants, aggregates, viruses, etc. Purity Remove bulk impurities Isolate product, concentrate, stabilize Polishing Intermediate purification Capture Step 7 Sample Preparation General considerations: • Select extraction procedure according to source and location of protein • Use gentle procedures to minimize acidification and release of proteolytic enzymes • Work quickly at sub-ambient temperatures • Use buffer to maintain pH, ionic strength Goal: To stabilize sample 8 Always Limit the Number of Steps Maximize the Yield at Each Step Yield (%) 100 80 95% / step 60 90% / step 40 85% / step 20 80% / step 75% / step 20% overall yield! 0 1 2 3 4 5 6 7 8 Number of steps 9 Gel Filtration Gel Filtration (GF) Chromatography The principle of gel filtration -- excluded volume [Note: gel filtration chromatography is also sometimes called “size exclusion chromatography”] Vo = “void volume” Vt = “bed volume” Ve = “elution volume” Vi = Vt - Vo Principles of gel chromatography (con’d) Gel Filtration Elution Volumes as a Function of Molecular Weight Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. Ion Exchange Chromatography Ion Exchange (IEX) Chromatography Ion Exchange Chromatography (con’d) Some other popular chromatographic methods: • Hydrophobic interaction chromatography • Affinity chromatography • Reverse phase chromatography Hydrophobic Interaction Chromatography (HIC) Affinity Chromatography “Reversed Phase” Chromatography (RPC) (elution with organic solvents) Linking Chromatography Techniques Start conditions Technique End conditions Small sample volume GF Diluted sample Buffer change (if required) Low ionic strength IEX High ionic strength or pH change High ionic strength HIC Low ionic strength Specific binding conditions AC Specific elution conditions 23 In addition, there are non-chromatographic protein purification techniques, e. g.: • Ammonium sulfate precipitation • Sedimentation (rare) • Recombinant gene product over-expression • Refractile body prep (see above) • Detergent extraction • Heat treatment (especially for recombinant thermophile proteins expressed in E. coli) • Etc. Once You’ve Purified Your Protein, How Do You Characterize It? Some typical analytical tests: - SDS PAGE (both reducing and non-reducing) - Bioassay (if you have one) - Total protein determination - UV spectrophotometry - CD spectrometry - disulfides? - amino acid analysis - N-terminal (& C-terminal?) sequencing - HPLC? - metal analysis - mass spectrometry - NMR spectrometry & X-ray crystallography* - other? *Not usually used for routine analytical purposes!! Protein Size Determination by SDS Polyacrylamide Gel Electrophoresis - electrode Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984 + electrode Circular Dichroism Spectroscopy of Polypeptides Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984