Chem4711%28NoteSet2%29

Chem4711%28NoteSet2%29 - Protein Structure and Function If...

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Protein Structure and Function If you want to study a protein, you first have to purify it. “Don’t waste clean thinking on dirty enzymes.” Arthur Kornberg, Nobel Prize laureate How to go from a complex mixture (cell extract) to one protein. May be 5,000-10,000 different proteins in a cell. Vary in size (50-3,000 A. A.) Vary in shape (globular vs. fibrous) Vary in charge Vary tremendously in stability Vary tremendously in ability to bind specific ligands
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All’s fair in love, war, and protein purification. Take advantage of any property to isolate your protein. If the protein has never been purified: Choose source with high concentration of protein. High “purity” at start simplifies purification. If the protein has been previously purified: Different tissue/species, similar protocol. Virus/prokaryote/eukaryote usually quite different.
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Clone the protein. A. Ideally, put the protein in E. coli - cheap, lots of protein, easy to purify. BUT - toxicity issues, size issues, codon usage issues, post- translational modification. B. For eukaryotic proteins, put in bacculovirus, grow virus in insect cells. Proteins express well, post translational modification, toxicity OK BUT - more expensive, slower, lower yield If protein not cloned, must have a way to detect it. Enzymes - perform a chemical reaction. Structural proteins - no chemistry. Must use some other property (spectra, ligand binding, antibody binding, etc.) During purification: Maximize yield Maximize purity [Your Protein] [Total Protein]
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Break open cells. Remove debris. Stabilize your protein. Oxidation Hydrophobicity issues Degradation of your protein by proteases Protein denaturing Think cold, think fast, maybe modify solvent Also need to think about long term storage. Now, purify protein!
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Protein purification - source Most common: overexpression in heterologous system - E.coli - insect cells (Sf9) - Chinese hamster ovary (CHO) cells More traditional: animal parts - bovine liver - bovine heart - human blood
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Proteins - ion exchange chromatography If cation exchange: very positive = binds tightly (need salt to elute) Neutral and negative run through (Stepwise vs gradient)
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Proteins - ion exchange chromatography Cation exchange: very positive proteins bind tightly (need salt to elute) neutral and negative run through i.e. SP-sepharose Anion exchange: very negative proteins bind tightly (need salt to elute) neutral and positive run through i.e. DEAE-sepharose; Q-sepharose Stepwise vs gradient elution
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Chem4711%28NoteSet2%29 - Protein Structure and Function If...

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