Name: a convenient and general expression platform for the production of secreted proteins from human cells
Uploaded: 2012-07-31T13:00:00
Description: Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

Эта работа была поддержана Онтарио лечению ВИЧ исследовательская сеть Операционная Грант (ROG-G645) и канадского Института исследований в области здравоохранения премию Новый следователь (MSH-113554) для JEL и Университет Торонто стипендии для HA, FCA, и Джойнт. Авторы хотели бы поблагодарить Марни Фуско, Дафна Абельсон и доктора Эрика Ollmann Saphire на Scripps Research Institute (Ла-Хойя, Калифорния) за предоставление клеток, ebolavirus GP вектор экспрессии и общие рекомендации.

1. Подготовительные работы - Конструкции и клеточных культур Перед началом протокола гена должны быть кодон оптимизированы для экспрессии в клетках млекопитающих, и клонируют в соответствующий вектор экспрессии с использованием стандартных методов молекулярной биолог...

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10-слойный заводов клетки являются эффективным судно для производства миллиграмм количества белка. Основное преимущество использования ячейки завода по сравнению с другими традиционными судами, такими как ролик бутылки, дрожание колбы или счетчика колбы, в том, что они не требуют прио...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Щелочной фосфатазы (BCIP / НБТ) жидкий раствор субстрата </td><td> Сигма </td><td> B6404 </td><td></td></tr><tr><td> Антибиотикам / противогрибковым, 100X </td><td> Invitrogen </td><td> 15240062 </td><td></td></tr><tr><td> Anti-HA близость матрицы, клон 3F10 </td><td> Roche </td><td> 1815016 </td><td></td></tr><tr><td> Anti-HA мышиных МКА, клон 16B12 </td><td> Covance </td><td> MMS-101P </td><td></td></tr><tr><td> Колба клеточной культуры, T75 см 2 ткани культуры рассматривается </td><td> Гранулирование </td><td> 430641 </td><td></td></tr><tr><td> Колба клеточной культуры, T225 см 2 ткани культуры рассматривается </td><td> Гранулирование </td><td> 431082 </td><td></td></tr><tr><td>Пластин клеточной культуры, 6-а культуры тканей лечение </td><td> Гранулирование </td><td> 3516 </td><td></td></tr><tr><td> Сотовые завод, 10-слой CellSTACK </td><td> Гранулирование </td><td> 3312 </td><td></td></tr><tr><td> Centramate Омега 5K кассеты </td><td> Надоедать </td><td> OS005C12 </td><td></td></tr><tr><td> Centramate Омега 30К кассеты </td><td> Надоедать </td><td> OS030C12 </td><td></td></tr><tr><td> Хроматографии стеклянную колонку, 1.0x10 см </td><td> Kontes </td><td> 4204001010 </td><td></td></tr><tr><td> Ципрофлоксацин </td><td> Сигма </td><td> 17850 </td><td></td></tr><tr><td> CO 2 </td><td></td><td></td><td></td></tr><tr><td> Дульбекко изменения Орла СМИ (DMEM) </td><td> Сигма </td><td> D5796 </td><td></td></tr><tr><td> Эмбриональной телячьей сыворотки (FBS), тепло инактивированная </td><td> Invitrogen </td><td> 12484-028 </td><td></td></tr><tr><td> FuGENE HD трансфекции реагент </td><td> Promega </td><td> 4709691001 </td><td></td></tr><tr><td> GeneJuice трансфекции реагент </td><td> EMD / Merck </td><td> 70967-6 </td><td></td></tr><tr><td> Глицин </td><td> Сигма </td><td> G8898 </td><td></td></tr><tr><td> Антимышиного IgG F (аb &#39;) 2 щелочные </td><td> Thermo Scientific </td><td> 31324 </td><td></td></tr><tr><td> фосфатазы, конъюгированных антител </td><td></td><td></td><td></td></tr><tr><td> Гемагглютинина (HA) пептид, 100 мг </td><td> Genscript </td><td> синтез новых </td><td></td></tr><tr><td> (Последовательность: YPYDVPDYA, 95% чистоты) </td><td></td><td></td><td></td></tr><tr><td> HEK 293T клеток </td><td> ATCC </td><td> CRL-11268 </td><td></td></tr><tr><td> Householг хлорной извести (4% вес / объем гипохлорита натрия) </td><td> различных марок доступны </td><td></td><td></td></tr><tr><td> Immobilon-P PVDF мембраны </td><td> Millipore </td><td> IPVH07850 </td><td></td></tr><tr><td> MiniPrep плазмиды комплект очистки, PureLink быстрого </td><td> Invitrogen </td><td> K2100-11 </td><td></td></tr><tr><td> MaxiPrep плазмиды комплект очистки, PureLink HiPure </td><td> Invitrogen </td><td> K2100-07 </td><td></td></tr><tr><td> NaN 3 </td><td> Сигма </td><td> S8032 </td><td></td></tr><tr><td> вектор pDISPLAY выражение </td><td> Invitrogen </td><td> V660-20 </td><td></td></tr><tr><td> Пенициллина / стрептомицина (ручка / стрептококк), 100X </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Фосфат-солевом буфере (PBS), стерильные 1X </td><td> Сигма </td><td> D8537 </td><td></td></tr><tr><td> Полиэтиленимина (PEI), линейные 25 кДа </td><td> Polyscience </td><td> 23966 </td><td></td></tr><tr><td> Обезжиренное молоко сухой порошок </td><td> Гвоздика </td><td></td><td></td></tr><tr><td> Stericup-GP PES блок фильтрации вакуума, </td><td> Millipore </td><td> SCGPU05RE </td><td></td></tr><tr><td> 0,22 мкм, емкостью 500 мл </td><td></td><td></td><td></td></tr><tr><td> Трипановый синий </td><td> Invitrogen </td><td> 15250061 </td><td></td></tr><tr><td> Трипсин-EDTA, 0.05% (вес / объем) </td><td> Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Твин-20 </td><td> Сигма </td><td> P7949 </td><td></td></tr><tr><td> Вальпроевая кислота </td><td> Сигма </td><td> P4543 </td><td></td></tr><tr><td> Centramate тангенциальных потоков в системе </td><td> Надоедать </td><td></td><td></td></tr><tr><td> CO 2 увлажненном инкубаторе, стандартный 6,0 куб. футов </td><td> различных марок доступны </td><td></td><td></td></tr><tr><td> Электрофорез и передачи единицы </td><td> различных марок доступны </td><td></td><td></td></tr><tr><td> Инкубатор, 37 ° C </td><td> различных марок доступны </td><td></td><td></td></tr></tbody></table>

a convenient and general expression platform for the production of secreted proteins from human cells

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.

Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

В пост-человеческой эры геномики, наличие рекомбинантных белков в родной конформации имеет решающее значение для структурных, функциональных и терапевтических исследований и разработок. Здесь мы описываем испытаний и масштабные экспрессии белков системы человеческих эмбриональных почек 293T клетки, которые могут быть использованы для производства различных рекомбинантных белков.

Удобный и общее выражение платформы для производства секретируемых белков из клеток человека

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Распространение человеческих эмбриональных стволовых (ЭС) клеток

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Подготовка первичных гемопоэтических клеточных культур из костного мозга мышей для Электропорация

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Окрашивание белков в гелях Кумасси G-250 без органических растворителей и уксусной кислоты

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Использование генной Pulser MXcell Электропорация системы для трансфекции первичных элементов с высоким КПД

The MultiBac Protein Complex Production Platform at the EMBL

MultiBac белкового комплекса добывающей платформы на EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Эффективное Агроинфильтрация растений для высокого уровня переходных Экспрессия рекомбинантных белков

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Эффективное производство и очистка рекомбинантного мышиного Kindlin-3 от клетках насекомых биофизической исследований

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Зеленый флуоресцентный белок на основе Expression Скрининг мембранных белков в<em&gt; Кишечная палочка</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

Обогащение белков внеклеточного матрикса из тканей и пищеварения в пептидов для анализа масс-спектрометрии

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

High Yield Экспрессия рекомбинантных белков человека с временной трансфекции клеток НЕК293 в виде суспензии

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...