मानव कोशिकाओं से secreted प्रोटीन के उत्पादन के लिए एक सुविधाजनक और जनरल अभिव्यक्ति प्लेटफार्म
Summary
बाद मानव जीनोमिक्स युग में, देशी रचना में पुनः संयोजक प्रोटीन की उपलब्धता संरचनात्मक, कार्यात्मक और चिकित्सकीय अनुसंधान और विकास के लिए महत्वपूर्ण है. यहाँ, हम एक परीक्षण और बड़े पैमाने पर मानव भ्रूण गुर्दे 293T कोशिकाओं है कि पुनः संयोजक प्रोटीन की एक किस्म का उत्पादन करने के लिए इस्तेमाल किया जा सकता है में प्रोटीन अभिव्यक्ति प्रणाली का वर्णन.
Abstract
Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.
Protocol
Discussion
10 परत सेल कारखानों मिलीग्राम प्रोटीन की मात्रा के उत्पादन के लिए एक प्रभावी पोत हैं. रोलर की बोतलें, हिला बोतल या स्पिनर बोतल के रूप में अन्य परंपरागत वाहिकाओं, पर सेल कारखाने का उपयोग करने का एक बड़ा फा?…
Declarações
The authors have nothing to disclose.
Acknowledgements
यह काम हा, FCA, और जे डी सी के लिए एक ओंटारियो एचआईवी उपचार नेटवर्क अनुसंधान ऑपरेटिंग (रोग – G645) अनुदान और स्वास्थ्य अनुसंधान अन्वेषक पुरस्कार (MSH 113,554) की कनाडा के संस्थानों jel करने के लिए, और टोरंटो विश्वविद्यालय की फैलोशिप द्वारा समर्थित किया गया. लेखकों के स्क्रिप्स रिसर्च इंस्टीट्यूट (ला Jolla, CA) में Marnie Fusco, Dafna Abelson और डॉ. एरिका Ollmann Saphire प्रदान कोशिकाओं, ebolavirus जीपी अभिव्यक्ति वेक्टर और सामान्य सलाह के लिए धन्यवाद करना चाहते हैं.
Materials
| Name of reagent | Company | Catalogue number | Comments |
| Alkaline phosphatase (BCIP/NBT) liquid substrate solution | Sigma | B6404 | |
| Antibiotic/Antimycotic, 100X | Invitrogen | 15240062 | |
| Anti-HA affinity matrix, clone 3F10 | Roche | 1815016 | |
| Anti-HA murine mAb, clone 16B12 | Covance | MMS-101P | |
| Cell culture flask, T75 cm2 tissue culture treated | Corning | 430641 | |
| Cell culture flask, T225 cm2 tissue culture treated | Corning | 431082 | |
| Cell culture plates,6-well tissue culture treated | Corning | 3516 | |
| Cell factory, 10-layer CellSTACK | Corning | 3312 | |
| Centramate Omega 5K Cassette | Pall | OS005C12 | |
| Centramate Omega 30K Cassette | Pall | OS030C12 | |
| Chromatography glass column, 1.0×10 cm | Kontes | 4204001010 | |
| Ciprofloxacin | Sigma | 17850 | |
| CO2 | |||
| Dulbecco’s modified Eagle’s media (DMEM) | Sigma | D5796 | |
| Fetal bovine serum (FBS), heat inactivated | Invitrogen | 12484-028 | |
| FuGENE HD transfection reagent | Promega | 4709691001 | |
| GeneJuice transfection reagent | EMD/Merck | 70967-6 | |
| Glycine | Sigma | G8898 | |
| Goat anti-mouse IgG F(ab’)2 alkaline | Thermo Scientific | 31324 | |
| phosphatase-conjugated antibody | |||
| Hemagglutinin (HA) peptide, 100 mg | Genscript | custom synthesis | |
| (sequence: YPYDVPDYA; 95% purity) | |||
| HEK 293T cells | ATCC | CRL-11268 | |
| Household bleach (4% w/v sodium hypochlorite) | various brands are available | ||
| Immobilon-P PVDF membrane | Millipore | IPVH07850 | |
| MiniPrep plasmid purification kit, PureLink Quick | Invitrogen | K2100-11 | |
| MaxiPrep plasmid purification kit, PureLink HiPure | Invitrogen | K2100-07 | |
| NaN3 | Sigma | S8032 | |
| pDISPLAY expression vector | Invitrogen | V660-20 | |
| Penicillin/streptomycin (pen/strep), 100X | Invitrogen | 15140-122 | |
| Phosphate-buffered saline (PBS), sterile 1X | Sigma | D8537 | |
| Polyethyleneimine (PEI), linear 25 kDa | Polyscience | 23966 | |
| Skim milk dry powder | Carnation | ||
| Stericup-GP PES vacuum filtration unit, | Millipore | SCGPU05RE | |
| 0.22 μm, 500 ml capacity | |||
| Trypan blue | Invitrogen | 15250061 | |
| Trypsin-EDTA, 0.05% (w/v) | Invitrogen | 25300-054 | |
| Tween-20 | Sigma | P7949 | |
| Valproic acid | Sigma | P4543 | |
| Centramate tangential flow system | Pall | ||
| CO2 humidified incubator, standard 6.0 cu. ft. | various brands are available | ||
| Electrophoresis and transfer unit | various brands are available | ||
| Incubator, 37 °C | various brands are available |
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