Protein purification workshop

2. Protein Purification: From industrial enzymes to cancer therapy

3. Protein Expression and Purification Series Instructors Jim DeKloe Solano Community College [email_address] Bio-Rad Curriculum and Training Specialists : Sherri Andrews, Ph.D. (Eastern US) [email_address] Leigh Brown, M.A. (Central US) [email_address] Damon Tighe (Western US) [email_address]

5. Protein Expression and Purification Series Option 1 Centrifugation Purification Module Option 3 Prepacked Cartridge Purification Module Option 2 Handpacked Column Purification Module Growth and Expression Module SDS-PAGE Electrophoresis Module DHFR Enzymatic Assay Module Purification Module

8. The Value of Proteins Price Per Gram *Prices in 2011 US Dollars $1,357,000 Granulocyte Colony Stimulating Factor $227,000 Growth Hormone $60 Insulin $48 Gold $14 Bovine Growth Hormone

9. Biomanufacturing Defined The production of pharmaceutical proteins using genetically engineered cells

11. Expression Choices Secreted Often secreted into media Intracellular Product Extracellular Intracellular or extracellular Intracellular Deposition of product Easy Easy Difficult Relative ease of recovery Difficult Easy Easy Relative ease to grow Full Yes, but different pattern No Glycosylation Yes Probably Sometimes Folding Low High High Product titer (concentration) High Low Low Cost of growth medium High Low Low Contamination risk Mammalian Yeast Bacteria Parameter

12. Protein – The product of Biotech Yeast Vaccination Human papillomavirus vaccine Yeast Vaccination Hepatitis B virus vaccine CHO cells Anemia Erythropoietin CHO cells Heart attack Tissue plasminogen activator Cancers Growth disorders Diabetes USED IN THE TREATMENT OF: E. Coli E. coli E. coli Cell Production Granulocyte colony stimulating factor Human growth hormone Insulin PROTEIN:

16. Induction Biotech companies genetically engineer plasmids to place genes behind inducible promoters

17. Transcriptional Regulation in the pDHFR system Lactose IPTG RNA Polymerase Z Y A Z Y A LacI Effector (Lactose) Z Y A LacI lac Operon

18. 2 phases of growth

23. Protein Expression and Purification Series Workflow Streak Cells Overnight culture Subculture, monitor, and induce Harvest and lyse cells Analyze Purify Centrifugation or Instrumentation

32. Size exclusion purification (buffer exchange) Eluate fraction GST-DHFR-His in 20 mM sodium phosphate, 300 mM NaCl and 250 mM imidazole Imidazole 250 mM imidazole solution has an A 280 = 0.2-0.4 W and Y contribute to A 280 of proteins NEED TO REMOVE IMIDAZOLE TO QUANTIFY PROTEIN CONCENTRATION USING A 280

33. Size Exclusion

34. Size exclusion purification (buffer exchange) Label desalting column with your initials. Prepare desalting column by inverting sharply several times to resuspend gel Centrifuge for 2 min at 1,000 x g . Discard remaining packing buffer and collection tube. Snap off tip and place in 2 ml collection tube. Remove green top cap. Allow excess packing buffer to drain by gravity to top of resin bed. If the column does not begin to flow, push the cap back on the column and then remove to start the flow. After draining, place column in clean 2 ml tube. Preparing the size exclusion column for usage

35. Size exclusion purification (buffer exchange) Eluate Desalted eluate Label new 2 ml tube Desalted eluate. Carefully apply 75 ul of eluate fraction directly to the center of column. Be careful not to touch resin with pipet tip. Centrifuge for 4 min at 1,000 x g . 75 µl Removing the 250 mM imidazole solution by size exclusion chromatography Collected fraction Desalted Eluate ~75 µl

36. Protein analysis (Quantitation using A 280 ) Desalted eluate Clean UV cuvette Set absorbance to 280 nm Blank spec with distilled H 2 O Measure absorbance of sample at 280nm Print out your data 75 µl

38. Enzyme Assay

39. Instrumentation BioLogic LP Demo BioLogic™ LP BioLogic DuoFlow™

40. Biomanufacturing Scaling up of the process developed during research and development