Purification of FLAG-tagged Secreted Proteins from Mammalian Cells
从哺乳动物细胞中纯化FLAG标记的分泌蛋白
发布: 2017年08月05日第7卷第15期 DOI: 10.21769/BioProtoc.2430 浏览次数: 11139
评审: Jyotiska ChaudhuriLiang LiuAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
31-Jan 2017
Abstract
This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays.
Keywords: Secreted protein (分泌蛋白)
Purification (纯化)
Mammalian (哺乳动物)
Ligand (配体)
Receptor (受体)
Background
E. coli is one of the organisms of choice for production of recombinant proteins from both prokaryotes and eukaryotes. The major advantages of bacterial expression systems are high productivity and low cost. However, mature proteins are essential for functional analyses (e.g., ligand binding assays). Most secreted eukaryotic proteins undergo post-translational modification by covalent glycan linkage and formation of disulfide bonds in the endoplasmic reticulum. These covalent modifications are essential for the general stability and folding of the secreted mature proteins. Therefore, the best method for production of mammalian secreted proteins is the use of a mammalian host to ensure the production of recombinant proteins that have undergone proper post-translational modifications. In the following protocol, we have described the detailed procedure for purification of secreted proteins from mammalian cells. This protocol is for production of FLAG-tagged proteins, but should be applicable to that of other tagged (e.g., His-tag) proteins as well.
Materials and Reagents
- Pipette tips (VWR)
- 12-well plate (Corning, Falcon®, catalog number: 353043 )
- 100 mm tissue culture dishes (Corning, Falcon®, catalog number: 353803 )
- Amicon Ultra 15 filtration units 3K (EMD Millipore, catalog number: UFC900308 )
- 1.5 ml tubes (VWR, catalog number: 89000-028 )
- 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
- 1 ml syringes (Terumo Medical, catalog number: SS-01T )
- 30 G needles (BD, catalog number: 305128 )
- Flp-In T-rex 293 cell line (Thermo Fisher Scientific, InvitrogenTM, catalog number: R78007 ) (Optional)
- pcDNA5 FRT TO (Thermo Fisher Scientific, InvitrogenTM, catalog number: V652020 ) (Optional)
- Hygromycin B (NACALAI TESQUE, catalog number: 07296-11 ) (Optional)
- Blasticidin (Wako Pure Chemical Industries, catalog number: 029-18701 ) (Optional)
- Doxycycline (Takara Bio, Clontech, catalog number: 631311 ) (Optional)
- Dulbecco’s modified eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
- Fetal bovine serum (FBS) (Biosera, catalog number: FB-1345/500 )
- Anti-Flag M2 agarose beads (Sigma-Aldrich, catalog number: A2220 )
- Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
- Potassium acetate (KAc) (Sigma-Aldrich, catalog number: 236497 )
- HEPES (Sigma-Aldrich, catalog number: H3375 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
- 3xFlag peptide (Sigma-Aldrich, catalog number: F4799 )
- Phosphate buffered saline (PBS)
- Growth medium (see Recipes)
- Wash buffer (see Recipes)
- Elution buffer (see Recipes)
Equipment
- Pipettes (e.g., Gilson)
- 37 °C, 5% CO2 incubator (e.g., Panasonic Biomedical, catalog number: MCO-170AICUV-PE )
- Tube rotator located in cold room (e.g., Fisher Scientific, catalog number: 88-861-049 )
- Centrifuge (e.g., Eppendorf, model: 5804 )
- Cell culture microscope (e.g., Carl Zeiss, Vienna, Austria)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Itakura, E., Chen, C. and de Bono, M. (2017). Purification of FLAG-tagged Secreted Proteins from Mammalian Cells. Bio-protocol 7(15): e2430. DOI: 10.21769/BioProtoc.2430.
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分类
生物化学 > 蛋白质 > 分离和纯化
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