1. Protein Purification Why Purify- Arthur Kornberg Handout Strategy –Starting materials, –Capture, Intermediate Purification, polishing Assays, quantitation and documentation

2. Protein Purification Principles I (From APBiotech Biochemistry 472/578 Resources) Biochemistry 472/578 Resources Define objectives –for purity, activity and quantity required of final product to avoid over or under developing a method Define properties of target protein and critical impurities –to simplify technique selection and optimisation Develop analytical assays –for fast detection of protein activity/recovery and to work efficiently Remove damaging contaminants early –for example, proteases

3. Protein Purification Principles II Use a different technique at each step –to take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity) Minimize sample handling at every stage –to avoid lengthy procedures which risk losing activity/reducing recovery Minimize use of additives –additives may need to be removed in an extra purification step or may interfere with activity assays Minimize number of steps - KEEP IT SIMPLE! –extra steps reduce yield and increase time, combine steps logically

4. Starting materials Natural source or artificial expression system Host for expression, –Bacteria, yeast, plants, transgenic animals Abundance, contaminants Lysis and clarification procedures –Native or denaturing conditions Subcellular fractionation Selective precipitation –PEI or Streptomycin Sulfate for RNA and DNA –Ammonium Sulfate for Proteins

5. Capture Quickly remove most damaging contaminants Concentrate, adsorption methods –Ion Exchange most general –Affinity chromatography can combine capture, intermediate and polishing steps –This step should remove most unwanted contaminants

6. Intermediate purification Use a different technique Affinity chromatography, Hydrophobic interaction chromatography Starting conditions are specific for each technique –Buffer must be compatible with adsorption –Can change buffer by dialysis or desalting by GFC Adsorption techniques result in small volume concentrated sample

7. Polishing Final removal of trace contaminants Often size exclusion chromatography –Buffer exchange is a part of the process –Sample volume always increases need to start with a concentrated sample Sample can be concentrated by –Precipitation (selective or nonselective) –Ultrafiltration (dialysis under pressure)

8. Assays, Quantitation and Documentation Assay enzyme activity at every step –Contaminants at early stages can mask or inhibit activity –Inactivation can occur at high temperatures, because of proteolysis, oxidation, aggregation, etc. Assay total protein Run an SDS gel to visualize specific contaminants Specific activity is defined as units of enzymatic activity per unit of total protein - Yeild can be defined in terms of total protein mass, and total enzyme units Goal is a high yield and high specific activity.

9. Expt 5 Purification of Alkaline Phosphatase (AP) Periplasmic Protein in E. coli –The space between the rigid peptidoglycan cell wall and the osmotically sensitive plasma membrane Phosphate scavenger –Liberates Pi from a variety of substrates –Induced by phosphate starvation Used to remove terminal phosphates for selective DNA ligation reactions Heat stable, Zn enzyme Will be used for enzyme kinetics in Experiment 6.

10. Text Book Purification Overview (N &B 175-195) 1. Lysozyme treatment to release periplasmic proteins –Centrifugation to separate soluble AP from cells –Dialysis to remove starting buffer (overnight) 2. Heat treatment to precipitate weaker proteins –Centrifugation to separate soluble AP from insoluble PPT –Ammonium sulfate to concentrate proteins/remove non protein contaminants Dialysis to remove ammonium sulfate (O/N) 3. Anion exchange (DEAE) chromatography –Step elution with 0.125M Salt 4. SDS Page to quantify the proteins in each fraction

11. Starting material E. coli cells starved for phosphate –Sucrose shrinks the plasma membrane reduces turgor pressure –Lysozyme cleave glycosidic linkages in cell wall –DNAse reduces viscosity from inadvertantly lysed cells –Left with AP, DNAse, Lysozyme, Sucrose other periplasmic and cytoplasmic contaminants

12. Alternative strategy Osmotic shock used to liberate periplasmic proteins –Many fewer proteins in periplasm than cytoplasm –Sucrose draws water from cytoplasm, shrinks inner membrane –EDTA permeabilizes cell wall –Transfer to low osmotic strength buffer causes the inner membrane to slam into the cell wall and force out periplasmic proteins –Periplasmic proteins, no lysozyme, no DNAase, not much sucrose –Periplasm rotococols in Biochemistry 472/578 Resources from NovagenBiochemistry 472/578 Resources

13. Assays Enzymatic assays –PNPP is hydrolyzed to PNP and Pi –Fixed time assay Mix enzyme and substrate, react for a fixed time, s top the reaction with a strong base, read the concentration of PNP at pH>10 –Continuous assay Monitor PNP production directly in the spec at ph 8 Bradford Assays for total protein SDS page for the distribution of proteins by size.