Application Note: ANCFGLYNXRPROTEIN 1012

Large Scale Recombinant Protein Production Using the Thermo Scientific Sorvall LYNX 6000 Superspeed Centrifuge and Fiberlite Large Capacity Rotors

Key Words Recombinant Protein Production, Proteomics, Large-Volume Pelleting, Fermentation, Transformation, Superspeed Centrifuges, Carbon Fiber Rotors

Introduction Large scale recombinant protein production is becoming increasingly important for applications in the field of proteomics. Protein characterization and functional studies can provide clues about the mode of action and can facilitate development of therapeutic drugs. For instance, using structural biology, inhibitors can be made that bind to, and inactivate, disease-related enzymes. Additionally, analysis of protein-DNA interactions enables scientists to better understand the expression of disease-related genes. In other cases, protein structure has aided in the study of antigen-antibody interaction and ligand-receptor binding, helping to understand immunology and molecular signal transduction. Figure 1. Thermo Scientific Sorvall LYNX 6000 superspeed However, to successfully study protein structure and centrifuge with Thermo Scientific Fiberlite F9-6x1000 LEX rotor. function, large quantities of protein (mg to g amounts) must be isolated from an appropriate expression Procedures system, such as a bacterial (e.g. Escherichia coli) or PROTOCOL 1: Basic Protocol for the Production of eukaryotic cell-based expression system (e.g. yeast, insect, Recombinant Protein in Bacterial Cells mammalian). In order to meet this need, cell cultures Protocol Adapted from Current Protocols in Protein Science, can be grown in large volume in a shake flask culture Bernard and Payton, 1995-1 or by the implementation of fermentation techniques. Using high cell density or fermentation, a researcher can 1. Inoculate 100 mL flask containing 20 mL LB medium optimize cell culture conditions to increase cell mass and with Escherichia coli (E. coli) expressing the gene of recombinant protein yield. interest. Shake overnight at 37 °C. The following protocols describe the usage of the Note: The protocol for the transformation of E. coli with a Thermo Scientific Sorvall LYNX 6000 superspeed gene of interest is detailed in protocol 3. Other methods centrifuge and large volume Thermo Scientific Fiberlite for the induction of gene expression in bacterial cells rotors to increase the efficiency of cell harvesting prior can be conducted using many commercially available to protein isolation. transfection kits. Materials and Methods During high volume cell culture, translated recombinant protein is secreted into the culture medium and/or remains intracellular. In either case, large scale centrifugation marks the first step in preparing the recombinant protein. Listed below are basic protocols for protein production in large volume bacterial cell cultures (up to 6 L). 2 A basic protocol for utilization of fermentation to produce results similar to those above is as follows (please follow manufacturer’s instructions for fermentation use): 1. Prepare the inocolum and fermentor. a. Prepare an overnight culture of transformed E. coli strain expressing the protein of interest. b. Grow culture until stationary phase is reached

(OD600 >1.5). Use overnight culture to inoculate the fermentor (volume is 1-10% of intended fermentation volume). 1 2 3 4 5 6 7 8 9 10 2. Sterilize the fermentor and medium. Figure 2. 4-20% SDS-PAGE of IL-8 NiNTA Purification. 20 mg of IL-8 was 3. Inoculate the fermentor, growth cells, and induce produced in a 4 L fermentation run, which is a ten-fold increase in production protein expression. over a previous shake flask culture of the same total volume. a. Inoculate fermentor by transferring inoculum 1. Bio-Rad® Prestained MW Marker 6. NiNTA Flow Through 3 aseptically into reactor vessel. 2. IL8 Standard (2 μg) 7. NiNTA Wash 1

3. NiNTA Load 8. NiNTA Wash 2 b. Monitor growth by measuring OD600 of samples at 4. NiNTA Flow Through 1 9. NiNTA Eluate 1 1 hr intervals. When target cell concentration is 5. NiNTA Flow Through 2 10. NiNTA Eluate 2 reached, proceed to induction. Target cell

concentration may be within an OD600 of 5 to 20. c. Induce protein expression via temperature or 2. Transfer overnight culture to 800 mL LB medium in a chemical induction. 3-L Erlenmeyer flask. Incubate and shake cells 1 to 2 hr at 37 °C. 4. Harvest the cells.

3. After cells reach exponential growth phase (OD600 of a. For small fermentors (1-10 L) 0.5 to 0.6), transfer cells to a 500 mL – 1 L chilled i. Pressurize the fermentor and transfer the reactor centrifuge bottle. contents into an intermediate container. 4. Place centrifuge bottles in the Thermo Scientific ii. Aliquot culture into 500 mL or 1 L centrifuge Fiberlite F9-6x1000 LEX, F10-4x1000 LEX, or bottles. F12-6x500 LEX rotor and perform centrifugation with the Sorvall® LYNX 6000 superspeed centrifuge with the iii. Perform centrifugation using the Sorvall LYNX following parameters: 4000 x g for 15 min at 4 °C. 6000 superspeed centrifuge and the Fiberlite® F9-6x1000 LEX, F10-4x1000 LEX, or 5. Resuspend the pellet with the appropriate medium for F12-6x500 LEX rotor with the following subsequent protein analysis. parameters: 5000 x g for 15 min at 4 °C. PROTOCOL 2: Utilization of Fermentation to 5. Store the harvested cells and clean the fermentor. Enhance Recombinant Protein Production Protocol Adapted from Current Protocols in Protein Science, a. Collect pellets from each centrifuge bottle and Bernard and Payton, 1995-2 Kelli Benge, Research place together in a heat-sealed plastic bag. Pellets Associate, Zyomyx, Inc., Hayward, CA 94545 may be frozen at -20 °C and use for subsequent In a recent recombinant human Interleukin-8 production protein analysis. run, our group achieved a two-fold increase in the cell PROTOCOL 3: Production of Recombinant Protein density of a 4 L batch culture as compared to a previous by Electroporation of E.coli cells shake flask experiment, and a ten-fold increase in rhIL-8 Adapted from Current Protocols in Protein Science, Bernard yield (Figure 2). and Payton, 1995-1

The fermentation timeline followed that of the shake 1. Prepare competent cells. flask experiment, and no nutrient additions were made to the media. The only differences in the experiments a. Inoculate 100 mL flask containing 20 mL LB medium were that the dissolved oxygen was controlled at 30% with a single colony of the E. coli host strain to be (vs. uncontrolled in the shake flask), and the temperature transformed. Shake overnight at 37 °C. was maintained at 32 °C instead of 37 °C during the b. Transfer overnight culture to 800 mL LB medium cell growth phase. To optimize culture conditions in in a 3 L Erlenmeyer flask. Incubate and shake cells future experiments, pH will be regulated and glucose 1 to 2 hr at 37 °C. will be added to the media. Optimizing these two c. After cells reach exponential growth phase (OD of parameters alone will increase cell density at least six-fold 600 (unpublished observations), and we expect a concomitant 0.5 to 0.6), transfer cells to a 500 mL to 1 L chilled increase in recombinant protein yield. centrifuge bottle. Application Note: ANCFGLYNXRPROTEIN 1012

d. Perform centrifugation with the Sorvall LYNX 6000 Conclusion 3 SPANISH thermoscientific.comsuperspeed centrifuge and the Fiberlite F9-6x1000 In order to produce large quantities of protein for LEX, F10-4x1000 LEX, or F12-6x500 LEX rotor proteomics work, cell cultures must be large volume © 2012 Thermo Fisher Scientific Inc. Reservados todos los derechos. Las otras marcas comerciales son propiedad de Thermo Fisher Scientific Inc. y sus filiales. Características,with the condicionesfollowing y precios parameters: están sujetos a posibles 4000 cambios. x g Algunos for 15productos min no están disponiblesor cell endense. todos los países.Fermentation Consulte los improves efficiency by detalles conat su at representante 4 °C. comercial local. increasing cell density and provides easier handling of large cultures (>4 L), since the culture is contained in Alemania e. Discard +49 6184 supernatant 90 6000 and resuspendFrancia +33 pellet 2 2803 in 2180 5-10 mL Rusia +7 812 703 42 15 Australia +61 39757 4300 Holanda +31 76 579 55 55 one vesselSuiza +41as compared 44 454 12 22 to handling several shake flasks. Austria of+43 ice 1 801 cold 40 0 water. Bring to a finalIndia +91 volume 22 6716 2200of 800 mL UK/Irlanda +44 870 609 9203 Pelleting of bacterial cells from a large volume culture Bélgicawith +32 53 ice-cold 73 42 41 water and performItalia centrifugation+39 02 95059 554 with USA/Canadá +1 866 984 3766 China +86 21 6865 4588 or Japón +81 45 453 9220 can be completed in the Fiberlite F9-6x1000 LEX, +86 10 the8419 following3588 parameters: 4000Nueva x Zelandag for 15 +64 min 9 980 at 6700 Otros países de Asia +852 2885 4613 F10-4x1000 LEX, or F12-6x500 LEX rotors in the España/Portugal4 ºC. Repeat +34 93 223 twice. 09 18 Paises Nórdicos/Bálticos/CIS Otros paises +49 6184 90 6000 +358 9 329 10200 Sorvallo +33LYNX 2 2803 6000 2180 superspeed centrifuge. The Fiberlite f. Resuspend pellet in ice-cold water to a final volume of carbon fiber rotors are lightweight, and thus easy to 50 mL and perform centrifugation in chilled handle, and can process 3-6 L of cell culture in a single 50 mL centrifuge or conical tubes with the Sorvall run. The time saved in centrifugation with these large thermoscientific.comLYNX 6000 superspeed centrifugeITALIAN and the Fiberlite volume rotors improves the efficiency of downstream

© 2012F20-12x50 Thermo Fisher Scientific LEX Inc. or Tutti F14-14x50cy i diritti riservati. Tutti gli rotoraltri marchi with sono dithe proprietà di Thermo Fisherprocessing Scientific Inc. eof delle recombinant sue filiali. Specifiche, proteins. condizionifollowing e tariffe possono parameters: subire variazioni. Non 4000 tutti i prodotti x g forsono disponibili15 min in tuttiat i 4paesi. ºC. Per maggiori dettagli consultare il rivenditore locale References Australiag. Discard +61 39757 supernatant, 4300 estimateIndia the +91volume 22 6716 of 2200 the 1. Bernard,Svizzera A. +41 and 44 454M. 12Payton 22 (1995-1). Selection of Austria +43 1 801 40 0 Italia +39 02 95059 554 UK/Irlanda +44 870 609 9203 Belgio pellet,+32 53 73 and 42 41 add an equal volumeOlanda of +31ice-cold 76 579 55water. 55 EscherichiaUSA/Canada coli +1 Expression866 984 3766 Systems. Current Protocols Cina +86 21 6865 4588 or Nuova Zelanda +64 9 980 6700 in Protein Science 5.2.1-5.2.18. +86h. 10 Vortex, 8419 3588 distribute 100 µL aliquotsEuropa intodel Nord/CIS prechilled Altre Nazioni Asiatiche +852 2885 4613 Francia1.5 +33 mL 2 2803 sterile 2180 microcentrifuge+358 tubes. 9 329 Cell 10200 aliquots Nazioni non in elenco +49 6184 90 6000 Germania +49 6184 90 6000 Russia +7 812 703 42 15 2. Bernard,o +33 2A. 2803 and 2180 M. Payton (1995-2). Fermentation Giapponemay +81 be 45 used453 9220 immediately or storedSpagna/Portogallo with glycerol +34 93 223 09 18 and Growth of Escherichia coli for Optimal Protein at -80 °C. Production. Current Protocols in Protein Science 5.3.1-5.3.18. 2. Transform competent cells. thermoscientific.com GERMAN a. Add 5 pg to 0.5 µg plasmid DNA to 100 µL of © 2012 Thermo Fisher Scientific Inc. Alle Rechte vorbehalten. Alle anderenWarenzeichen sind Eigentum von Thermo Fisher Scientific Inc. bzw. nationalen Niederlassungen.competent Änderungen cells. von Spezifikationen, Mix by invertingBedingungen und tubes Preisen vorbehalten.several Nichttimes. alle Produkte sind in allen Ländern erhältlich. Ausführliche Informationen erhalten Sie bei Ihrer Thermo Fisher Vertretung. b. Set electroporation apparatus to 2.5 kV, 25 µF, 200 Ω. Australien +61 39757 4300 Indien +91 22 6716 2200 Russland +7 812 703 42 15 Belgienc. Transfer +32 53 73 42DNA 41 and cells to prechilledItalien +39 02electroporation 95059 554 Spanien/Portugal +34 93 223 09 18 China cuvette,+86 21 6865 place 4588 or cuvette into electroporationJapan +81 45 453 chamber 9220 Schweiz +41 44 454 12 22 +86 10 8419 3588 Niederlande +31 76 579 55 55 UK/Irland +44 870 609 9203 Deutschlandand apply Nationale the kostenlose electrical Hotline pulse. Neuseeland +64 9 980 6700 USA/Kanada +1 866 984 3766 0800 1 536 376 Nordeuropa/Baltikum/CIS Deutschlandd. Remove International cuvette, add 1 mL SOC+358 medium 9 329 10200 and Andere asiatische Länder +852 2885 4613 +49 6184transfer 90 6000 contents to a 20 mL sterileÖsterreich culture +43 1 801 tube. 40 0 Andere Länder +49 6184 90 6000 Frankreich +33 2 2803 2180 oder +33 2 2803 2180 Incubate 60 min with moderate shaking. e. Plate transformation culture (10 to 100 µL) onto LB plates to isolate single colonies. Single colonies can thermoscientific.comthen be picked to produce pureFRENCH and viable cultures of © 2012the Thermo bacteria Fisher Scientific expressing Inc. Tous droits yourréservés. protein Les autres marquesof interest. déposées sont des marques commerciales ou déposées de Thermo Fisher Scientific Inc et de ses filiales. Les caractéristiques, conditions et tarifs sont susceptibles d’être modifiés. Tous les produits ne sont pas disponibles dans tous les pays. Pour tout renseignement, veuillez vous adresser à votre distributeur local.

Allemagne +49 6184 90 6000 France +33 2 2803 2180 Russie +7 812 703 42 15 Australie +61 39757 4300 Inde +91 22 6716 2200 Suisse +41 44 454 12 22 Autriche +43 1 801 40 0 Italie +39 02 95059 554 UK/Irlande +44 870 609 9203 Belgique +32 53 73 42 41 Japon +81 45 453 9220 USA/Canada +1 866 984 3766 Chine +86 21 6865 4588 or Nouvelle-Zélande +64 9 980 6700 +86 10 8419 3588 Pays Bas +31 76 579 55 55 Autres pays d’Asie +852 2885 4613 Espagne/Portugal +34 93 223 09 18 Pays nordiques/baltes/CEI Autres Pays +49 6184 90 6000 +358 9 329 10200 ou +33 2 2803 2180

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Australia +61 39757 4300 India toll free 1800 22 8374 Russia +7 812 703 42 15 Austria +43 1 801 40 0 India +91 22 6716 2200 Spain/Portugal +34 93 223 09 18 Belgium +32 53 73 42 41 Italy +39 02 95059 554 Switzerland +41 44 454 12 22 China +86 21 6865 4588 or Japan +81 45 453 9220 UK/Ireland +44 870 609 9203 +86 10 8419 3588 Netherlands +31 76 579 55 55 USA/Canada +1 866 984 3766 France +33 2 2803 2180 New Zealand +64 9 980 6700 Germany national toll free 0800 1 536 376 Nordic/Baltic/CIS countries Other Asian countries +852 2885 4613 Germany international +49 6184 90 6000 +358 9 329 10200 Countries not listed +49 6184 90 6000

ANCFGLYNXRPROTEIN 1012