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. However, to successfully study protein structure and function, large quantities of protein (mg to g amounts) must be isolated from an appropriate expression system, such as a bacterial (e.g. Escherichia coli) or eukaryotic cell-based expression system (e.g. yeast, insect, mammalian). In order to meet this need, cell cultures can be grown in large volume in a shake flask culture or by the implementation of fermentation techniques. Using high cell density or fermentation, a researcher can optimize cell culture conditions to increase cell mass and recombinant protein yield. The following protocols describe the usage of the Thermo Scientific Sorvall LYNX 6000 superspeed centrifuge and large volume Thermo Scientific Fiberlite rotors to increase the efficiency of cell harvesting prior to protein isolation.
Figure 1. Thermo Scientific Sorvall LYNX 6000 superspeed centrifuge with Thermo Scientific Fiberlite F9-6x1000 LEX rotor.

Procedures
PROTOCOL 1: Basic Protocol for the Production of Recombinant Protein in Bacterial Cells
Protocol Adapted from Current Protocols in Protein Science, Bernard and Payton, 1995-1

1. Inoculate 100 mL flask containing 20 mL LB medium with Escherichia coli (E. coli) expressing the gene of interest. Shake overnight at 37 C. Note: The protocol for the transformation of E. coli with a gene of interest is detailed in protocol 3. Other methods for the induction of gene expression in bacterial cells can be conducted using many commercially available 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).

A basic protocol for utilization of fermentation to produce results similar to those above is as follows (please follow manufacturers 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).

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2. Sterilize the fermentor and medium. 3. Inoculate the fermentor, growth cells, and induce protein expression. a. Inoculate fermentor by transferring inoculum aseptically into reactor vessel. b. Monitor growth by measuring OD600 of samples at 1 hr intervals. When target cell concentration is reached, proceed to induction. Target cell concentration may be within an OD600 of 5 to 20. c. Induce protein expression via temperature or chemical induction. 4. Harvest the cells. a. For small fermentors (1-10 L) i. Pressurize the fermentor and transfer the reactor contents into an intermediate container. ii. Aliquot culture into 500 mL or 1 L centrifuge bottles. iii. Perform centrifugation using the Sorvall LYNX 6000 superspeed centrifuge and the Fiberlite F9-6x1000 LEX, F10-4x1000 LEX, or F12-6x500 LEX rotor with the following parameters: 5000 x g for 15 min at 4 C. 5. Store the harvested cells and clean the fermentor. a. Collect pellets from each centrifuge bottle and place together in a heat-sealed plastic bag. Pellets may be frozen at -20 C and use for subsequent protein analysis. PROTOCOL 3: Production of Recombinant Protein by Electroporation of E.coli cells
Adapted from Current Protocols in Protein Science, Bernard and Payton, 1995-1

Figure 2. 4-20% SDS-PAGE of IL-8 NiNTA Purification. 20 mg of IL-8 was produced in a 4 L fermentation run, which is a ten-fold increase in production over a previous shake flask culture of the same total volume. 1. 2. 3. 4. 5. Bio-Rad Prestained MW Marker IL8 Standard (2 g) NiNTA Load NiNTA Flow Through 1 NiNTA Flow Through 2 6. 7. 8. 9. 10. NiNTA Flow Through 3 NiNTA Wash 1 NiNTA Wash 2 NiNTA Eluate 1 NiNTA Eluate 2

2. Transfer overnight culture to 800 mL LB medium in a 3-L Erlenmeyer flask. Incubate and shake cells 1 to 2 hr at 37 C. 3. After cells reach exponential growth phase (OD600 of 0.5 to 0.6), transfer cells to a 500 mL 1 L chilled centrifuge bottle. 4. Place centrifuge bottles in the Thermo Scientific Fiberlite F9-6x1000 LEX, F10-4x1000 LEX, or F12-6x500 LEX rotor and perform centrifugation with the Sorvall LYNX 6000 superspeed centrifuge with the following parameters: 4000 x g for 15 min at 4 C. 5. Resuspend the pellet with the appropriate medium for subsequent protein analysis. PROTOCOL 2: Utilization of Fermentation to Enhance Recombinant Protein Production
Protocol Adapted from Current Protocols in Protein Science, Bernard and Payton, 1995-2 Kelli Benge, Research Associate, Zyomyx, Inc., Hayward, CA 94545

In a recent recombinant human Interleukin-8 production run, our group achieved a two-fold increase in the cell density of a 4 L batch culture as compared to a previous shake flask experiment, and a ten-fold increase in rhIL-8 yield (Figure 2). The fermentation timeline followed that of the shake flask experiment, and no nutrient additions were made to the media. The only differences in the experiments were that the dissolved oxygen was controlled at 30% (vs. uncontrolled in the shake flask), and the temperature was maintained at 32 C instead of 37 C during the cell growth phase. To optimize culture conditions in future experiments, pH will be regulated and glucose will be added to the media. Optimizing these two parameters alone will increase cell density at least six-fold (unpublished observations), and we expect a concomitant increase in recombinant protein yield.

1. Prepare competent cells. a. Inoculate 100 mL flask containing 20 mL LB medium with a single colony of the E. coli host strain to be transformed. Shake overnight at 37 C. b. Transfer overnight culture to 800 mL LB medium in a 3 L Erlenmeyer flask. Incubate and shake cells 1 to 2 hr at 37 C. c. After cells reach exponential growth phase (OD600 of 0.5 to 0.6), transfer cells to a 500 mL to 1 L chilled centrifuge bottle.

Application Note: ANCFGLYNXRPROTEIN 1012

d. Perform centrifugation with the Sorvall LYNX 6000 Conclusion SPANISH superspeed centrifuge and the Fiberlite F9-6x1000 In order to produce large quantities of protein for thermoscientific.com 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 with the following parameters: 4000 x g for productos no estn disponibles endense.pases. Consulte los 15 min or cell todos los Fermentation improves efficiency by filiales. Caractersticas, condiciones y precios estn sujetos a posibles cambios. Algunos detalles con su representante comercial local. at at 4 C. increasing cell density and provides easier handling of large cultures812 703 42 15 Alemania +49 6184supernatant and resuspend pellet in 5-10 mL Francia +33 2 2803 2180 Rusia +7 (>4 L), since the culture is contained in e. Discard 90 6000 Australia +61 39757 4300 Holanda +31 76 579 55 55 Suiza as 44 454 12 22 one vessel +41compared to handling several shake flasks. Austria of ice cold0water. Bring to a final +91 22 6716 2200 mL +43 1 801 40 India volume of 800 UK/Irlanda +44 870 609 9203 Pelleting of bacterial 984 3766 Blgicawith ice-cold water and perform centrifugation with +32 53 73 42 41 Italia +39 02 95059 554 USA/Canad +1 866 cells from a large volume culture China +86 21 6865 4588 or Japn +81 45 453 9220 can be completed in the Fiberlite F9-6x1000 LEX, +86 10 the following parameters: 4000 x Zelanda15 min at 8419 3588 Nueva g for +64 9 980 6700 Otros pases de Asia +852 2885 4613 F10-4x1000 LEX, 6184F12-6x500 LEX rotors in the Espaa/Portugal +34 93 223 09 18 Paises Nrdicos/Blticos/CIS Otros paises +49 or 90 6000 4 C. Repeat twice. +358 9 329 10200 o LYNX 6000 Sorvall +33 2 2803 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 ITALIAN thermoscientific.com superspeed centrifuge and the Fiberlite LYNX 6000 volume rotors improves the efficiency of downstream F20-12x50 LEX or F14-14x50cy altri marchi sono dithe di Thermo Fisher Scientific Inc. e delle recombinant proteins. rotor with propriet processing of sue filiali. Specifiche, 2012 Thermo Fisher Scientific Inc. Tutti i diritti riservati. Tutti gli following parameters: 4000 x g sono 15 min at paesi. Per condizioni e tariffe possono subire variazioni. Non tutti i prodottifor disponibili in tutti i4 C. maggiori dettagli consultare il rivenditore locale

References

Australia +61 39757 4300 g. Discard supernatant, estimateIndia +91 22 6716 2200 the volume of the Austria +43 1 801 40 0 Italia +39 02 95059 554 pellet,73 42 41 and add an equal volume of +31 76 579 55 55 Belgio +32 53 Olanda ice-cold water. Cina +86 21 6865 4588 or Nuova Zelanda +64 9 980 6700 h. 8419 3588 +86 10Vortex, distribute 100 L aliquots into prechilled Europa del Nord/CIS Francia +33mL sterile microcentrifuge tubes. Cell aliquots +358 9 329 10200 1.5 2 2803 2180 Germania +49 6184 90 6000 Russia +7 812 703 42 15 may be used immediately or stored with glycerol 223 09 18 Giappone +81 45 453 9220 Spagna/Portogallo +34 93

Svizzera and M. Payton (1995-1). Selection of 1. Bernard, A.+41 44 454 12 22 UK/Irlanda +44 870 609 9203 Escherichia coli Expression Systems. Current Protocols USA/Canada +1 866 984 3766 in Protein Science 5.2.1-5.2.18. Nazioni 2. Bernard, 2 non in elenco Payton 90 6000 A. and M. +49 6184 (1995-2). Fermentation o +33 2803 2180 and Growth of Escherichia coli for Optimal Protein Production. Current Protocols in Protein Science 5.3.1-5.3.18. Altre Nazioni Asiatiche +852 2885 4613

at -80 C. 2. Transform competent cells.

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GERMAN

a. Add 5 pg to 0.5 g plasmid DNA to 100 L of 2012 Thermo Fisher Scientific Inc. Alle Rechte vorbehalten. Alle anderenWarenzeichen sind Eigentum von Thermo Fisher Scientific Inc. bzw. nationalen competent cells. Mix by inverting tubes several times. Niederlassungen. nderungen von Spezifikationen, Bedingungen und Preisen vorbehalten. Nicht alle Produkte sind in allen Lndern erhltlich. Ausfhrliche
Informationen erhalten Sie bei Ihrer Thermo Fisher Vertretung.

b. Set electroporation apparatus to 2.5 kV, 25 F, 200 .

Russland +7 812 703 42 15 Spanien/Portugal +34 93 223 09 18 Schweiz +41 44 454 12 22 UK/Irland +44 870 609 9203 USA/Kanada +1 866 984 3766 Andere asiatische Lnder +852 2885 4613 Andere Lnder +49 6184 90 6000 oder +33 2 2803 2180

Australien +61 39757 4300 Indien +91 c. Transfer DNA and cells to prechilled 22 6716 2200 electroporation Belgien +32 53 73 42 41 Italien +39 02 95059 554 China cuvette, placeor +86 21 6865 4588 cuvette into electroporation chamber Japan +81 45 453 9220 +86 10 8419 3588 +31 76 579 55 and apply the electrical pulse. Niederlande +64 9 980 670055 Deutschland Nationale kostenlose Hotline Neuseeland 0800 1 536 376 Nordeuropa/Baltikum/CIS d. Remove cuvette, add 1 mL SOC medium and Deutschland International +358 9 329 10200 +49 6184 90 6000 contents to a 20 mL sterile culture tube. sterreich +43 1 801 40 0 transfer Frankreich +33 2 2803 2180

Incubate 60 min with moderate shaking.

e. Plate transformation culture (10 to 100 L) onto LB plates to isolate single colonies. Single colonies can FRENCH thermoscientific.com then be picked to produce pure and viable cultures of 2012the bacteria expressing rservs. protein of interest. des marques commerciales ou dposes de Thermo Fisher Thermo Fisher Scientific Inc. Tous droits your Les autres marques dposes sont
Scientific Inc et de ses filiales. Les caractristiques, conditions et tarifs sont susceptibles dtre modifis. Tous les produits ne sont pas disponibles dans tous les pays. Pour tout renseignement, veuillez vous adresser votre distributeur local.

Allemagne +49 6184 90 6000 Australie +61 39757 4300 Autriche +43 1 801 40 0 Belgique +32 53 73 42 41 Chine +86 21 6865 4588 or +86 10 8419 3588 Espagne/Portugal +34 93 223 09 18

France +33 2 2803 2180 Inde +91 22 6716 2200 Italie +39 02 95059 554 Japon +81 45 453 9220 Nouvelle-Zlande +64 9 980 6700 Pays Bas +31 76 579 55 55 Pays nordiques/baltes/CEI +358 9 329 10200

Russie +7 812 703 42 15 Suisse +41 44 454 12 22 UK/Irlande +44 870 609 9203 USA/Canada +1 866 984 3766 Autres pays dAsie +852 2885 4613 Autres Pays +49 6184 90 6000 ou +33 2 2803 2180

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