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   

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本实验方案简略版
Nature
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

  1. Pipette tips (VWR)
  2. 12-well plate (Corning, Falcon®, catalog number: 353043 )
  3. 100 mm tissue culture dishes (Corning, Falcon®, catalog number: 353803 )
  4. Amicon Ultra 15 filtration units 3K (EMD Millipore, catalog number: UFC900308 )
  5. 1.5 ml tubes (VWR, catalog number: 89000-028 )
  6. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  7. 1 ml syringes (Terumo Medical, catalog number: SS-01T )
  8. 30 G needles (BD, catalog number: 305128 )
  9. Flp-In T-rex 293 cell line (Thermo Fisher Scientific, InvitrogenTM, catalog number: R78007 ) (Optional)
  10. pcDNA5 FRT TO (Thermo Fisher Scientific, InvitrogenTM, catalog number: V652020 ) (Optional)
  11. Hygromycin B (NACALAI TESQUE, catalog number: 07296-11 ) (Optional)
  12. Blasticidin (Wako Pure Chemical Industries, catalog number: 029-18701 ) (Optional)
  13. Doxycycline (Takara Bio, Clontech, catalog number: 631311 ) (Optional)
  14. Dulbecco’s modified eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
  15. Fetal bovine serum (FBS) (Biosera, catalog number: FB-1345/500 )
  16. Anti-Flag M2 agarose beads (Sigma-Aldrich, catalog number: A2220 )
  17. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
  18. Potassium acetate (KAc) (Sigma-Aldrich, catalog number: 236497 )
  19. HEPES (Sigma-Aldrich, catalog number: H3375 )
  20. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  21. 3xFlag peptide (Sigma-Aldrich, catalog number: F4799 )
  22. Phosphate buffered saline (PBS)
  23. Growth medium (see Recipes)
  24. Wash buffer (see Recipes)
  25. Elution buffer (see Recipes)

Equipment

  1. Pipettes (e.g., Gilson)
  2. 37 °C, 5% CO2 incubator (e.g., Panasonic Biomedical, catalog number: MCO-170AICUV-PE )
  3. Tube rotator located in cold room (e.g., Fisher Scientific, catalog number: 88-861-049 )
  4. Centrifuge (e.g., Eppendorf, model: 5804 )
  5. Cell culture microscope (e.g., Carl Zeiss, Vienna, Austria)

Procedure

  1. Generation of stable cell lines using the Flp-In T-rex 293 cell line (Figure 1)
    Note: The Flp-In T-rex 293 cell line is suitable for inducible, stable, and homogenous high expression of your 3xFLAG-tagged protein of interest. Transiently transfected cell line can be used as long as the transfection efficiency (determined by immunofluorescence) is more than 50%.


    Figure 1. Diagram of how to prepare stable Flp-in T-rex 293 cell lines. Transfect Flp-in T-rex 293 cells with plasmids (pcDNA5 FRT TO with gene of interest and pOG44 that contains a gene for flippase). The following day, the cells are cultured with hygromycin B to select cells that have incorporated the gene of interest at FRT site of genomic region. The polyclonal stable cell lines are homogeneous expression.

    1. Seed 1 x 105 Flp-In T-rex 293 cells in each well of a 12-well plate.
    2. Incubate overnight when cells have reached approximately 50% of confluency, and transfect Flp-In T-rex 293 cells with pcDNA5 FRT TO containing your FLAG-tagged gene of interest and pOG44 plasmids.
    3. After 4-5 days of transfection, treat cells with 100 μg/ml hygromycin B and 15 μg/ml blasticidin for approximately 2 weeks.

  2. Purification of FLAG-tagged protein
    1. Grow five 100 mm dishes of Flp-In T-rex 293 cells expressing the 3xFLAG-tagged protein in growth medium (see Recipes) supplemented with 10 μg/ml doxycycline for 1 day until cells are approximately 80% confluent.
    2. Change cell culture media to 10 ml DMEM containing 5% FBS with 10 μg/ml doxycycline and culture for 2 days.
      Note: 10% FBS medium may clog an Amicon centrifuge filter during the concentration step.
    3. Harvest the cell culture media (conditioned media) from all five 100 mm dishes of step B2 into a 50 ml Falcon tube.
    4. Centrifuge the conditioned media at 2,000 x g for 5 min at 4 °C.
    5. Transfer the supernatant into an Amicon centrifuge filter unit. Spin at 4,000 x g for 30-40 min.
    6. Repeat step B5 until the retention volume is less than 2 ml.
    7. Transfer the concentrated conditioned media into two 1.5 ml tubes (1 ml per tube).
    8. Add 100 μl pre-washed 50% FLAG M2 beads slurry to each tube.
    9. Incubate the beads at least 2 h while rotating at 4 °C.
    10. Wash the beads with 0.5 ml cold wash buffer (see Recipes) 4 times by spinning at 100 x g for 1 min. After the last wash, remove as much buffer as possible with minimal disruption of the beads using syringe needles.
    11. Incubate the beads with 0.2 ml elution buffer (see Recipes) for 1 h at room temperature and collect the purified FLAG-tagged protein after centrifugation.

Recipes

  1. Growth medium (560.5 ml)
    500 ml DMEM
    55 ml 10% FBS
    5.5 ml 1% penicillin-streptomycin
  2. Wash buffer (50 ml)
    5.5 ml 1 M KAc (110 mM)
    1 ml 1 M HEPES (pH 7.4) (20 mM)
    100 μl 1 M MgCl2 (2 mM)
    Make up to 50 ml with Millipore water
  3. Elution buffer (210 μl)
    10 μl 5 mg/ml 3xFlag peptide (0.25 mg/ml)
    200 μl 1x PBS

Acknowledgments

This protocol was adapted from Chen et al. (2017). This work was supported by JSPS KAKENHI (Young Scientists A) Grant Number 16H06167, MEXT KAKENHI (Scientific Research on Innovative Areas) Grant Number 16H01194 (to E. I.); a European Molecular Biology Organization Fellowship (to C.C.); the Medical Research Council, UK; and the European Research Council (Advanced Grant 269058) grant to M.d.B.

References

  1. Chen, C., Itakura, E., Nelson, G. M., Sheng, M., Laurent, P., Fenk, L. A., Butcher, R. A., Hegde, R. S. and de Bono, M. (2017). IL-17 is a neuromodulator of Caenorhabditis elegans sensory responses. Nature 542(7639): 43-48.

简介

该方案描述了一种用于从哺乳动物细胞中用二硫键纯化糖基化的FLAG标记的分泌蛋白的方法。 纯化的产物可用于各种应用,例如配体结合测定。
【背景】电子。大肠杆菌是从原核生物和真核生物生产重组蛋白质的首选生物之一。细菌表达系统的主要优点是生产率高,成本低。然而,成熟蛋白质对于功能分析是必需的(例如,配体结合测定)。大多数分泌的真核蛋白通过共价聚糖键和在内质网中形成二硫键进行翻译后修饰。这些共价修饰对于分泌的成熟蛋白的一般稳定性和折叠是必需的。因此,生产哺乳动物分泌蛋白质的最佳方法是使用哺乳动物宿主来确保经过适当的翻译后修饰的重组蛋白的产生。在以下方案中,我们已经描述了从哺乳动物细胞中纯化分泌蛋白质的详细程序。该方案用于生产标记有FLAG标签的蛋白质,但也适用于其他标记(例如,His标签)蛋白质的蛋白质。

关键字:分泌蛋白, 纯化, 哺乳动物, 配体, 受体

材料和试剂

  1. 移液器吸头(VWR)
  2. 12孔板(Corning,Falcon ®,目录号:353043)
  3. 100毫米组织培养皿(康宁,Falcon ®,目录号:353803)
  4. Amicon Ultra 15过滤装置3K(EMD Millipore,目录号:UFC900308)
  5. (VWR,目录号:89000-028)
  6. 50ml Falcon管(Corning,Falcon ®,目录号:352070)
  7. 1 ml注射器(Terumo Medical,目录号:SS-01T)
  8. 30 G针(BD,目录号:305128)
  9. Flp-In T-rex 293细胞系(Thermo Fisher Scientific,Invitrogen TM,目录号:R78007)(可选)
  10. pcDNA5 FRT TO(Thermo Fisher Scientific,Invitrogen TM,目录号:V652020)(可选)
  11. 潮霉素B(NACALAI TESQUE,目录号:07296-11)(可选)
  12. 杀稻瘟菌素(Wako Pure Chemical Industries,目录号:029-18701)(可选)
  13. 多西环素(Takara Bio,Clontech,目录号:631311)(可选)
  14. Dulbecco改良的老鹰培养基(DMEM)(Sigma-Aldrich,目录号:D6546)
  15. 胎牛血清(FBS)(Biosera,目录号:FB-1345/500)
  16. 抗Flag M2琼脂糖珠(Sigma-Aldrich,目录号:A2220)
  17. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
  18. 乙酸钾(KAc)(Sigma-Aldrich,目录号:236497)
  19. HEPES(Sigma-Aldrich,目录号:H3375)
  20. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  21. 3xFlag肽(Sigma-Aldrich,目录号:F4799)
  22. 磷酸盐缓冲盐水(PBS)
  23. 生长培养基(见食谱)
  24. 洗涤缓冲液(见配方)
  25. 洗脱缓冲液(见配方)

设备

  1. 移液器(例如,,Gilson)
  2. 37℃,5%CO 2培养箱(例如,Panasonic Biomedical,目录号:MCO-170AICUV-PE)
  3. 位于冷室中的管旋转器(例如,Fisher Scientific,目录号:88-861-049)
  4. 离心机(例如,,Eppendorf,型号:5804)
  5. 细胞培养显微镜(例如,,Carl Zeiss,Vienna,Austria)

程序

  1. 使用Flp-In T-rex 293细胞系生成稳定的细胞系(图1)
    注意:Flp-In T-rex 293细胞系适用于您感兴趣的3xFLAG标记的蛋白质的诱导型,稳定和均匀的高表达。只要转染效率(通过免疫荧光测定)超过50%,可以使用瞬时转染的细胞系。


    图1.如何制备稳定的Flp-in T-rex 293细胞系图。 用质粒转染Flp-in T-rex 293细胞(pcDNA5 FRT TO与感兴趣的基因和含有翻转酶基因的pOG44)。第二天,用潮霉素B培养细胞以选择在基因组区域的FRT位点掺入感兴趣的基因的细胞。多克隆稳定细胞系是均一的表达
    1. 12孔板各孔中的1×10 5个Flp-In T-rex 293细胞。
    2. 当细胞达到约50%的融合时孵育过夜,并转染具有pcDNA5 FRT TO的Flp-In T-rex 293细胞,其含有您的FLAG标记的基因和pOG44质粒。
    3. 转染4-5天后,用100μg/ ml潮霉素B和15μg/ ml杀稻瘟素处理细胞约2周。

  2. FLAG标记蛋白的纯化
    1. 在补充有10μg/ ml多西环素的生长培养基(见食谱)中培养长达五个100毫米的Flp-In T-rex 293细胞的Flp-In T-rex 293细胞,表达3xFLAG标记的蛋白质,直到细胞约80%汇合。
    2. 将细胞培养基更换为含有10μg/ ml多西环素的含有5%FBS的10ml DMEM,培养2天。
      注意:在浓缩步骤中,10%FBS培养基可能会堵塞Amicon离心机过滤器。
    3. 将细胞培养基(条件培养基)从步骤B2的所有五个100mm的培养皿中收获到50ml的Falcon管中。
    4. 将条件培养基以2,000 x g离心5分钟,4℃
    5. 将上清液转移到Amicon离心机过滤器单元中。旋转4,000 x g 30-40分钟。
    6. 重复步骤B5直到保留体积小于2毫升。
    7. 将浓缩的条件培养基转移到两个1.5ml管中(每管1ml)
    8. 向每个管中加入100μl预洗的50%FLAG M2珠浆料。
    9. 在4℃下旋转时,至少孵育2小时。
    10. 用0.5ml冷洗涤缓冲液洗涤珠子(参见食谱)4次,以100×g旋转1分钟。最后一次洗涤后,尽可能多地使用缓冲液,用注射器针头尽可能少地破坏珠粒。
    11. 用0.2ml洗脱缓冲液(见食谱)将小珠孵育1小时,并离心后收集纯化的FLAG标记的蛋白质。

食谱

  1. 生长培养基(560.5ml)
    500毫升DMEM
    55毫升10%FBS
    5.5 ml 1%青霉素 - 链霉素
  2. 洗涤缓冲液(50ml)
    5.5 ml 1 M KAc(110 mM)
    1 ml 1 H HEPES(pH 7.4)(20 mM)
    100μl1M MgCl 2(2mM)
    用Millipore水补足50毫升
  3. 洗脱缓冲液(210μl)
    10μl5mg / ml 3xFlag肽(0.25mg / ml)
    200μl1x PBS

致谢

该方案由Chen等人改编(2017)。这项工作得到JSPS KAKENHI(Young Scientists A)授权号16H06167,MEXT KAKENHI(创新地区科学研究)授权号16H01194(授予E.I.)的支持。欧洲分子生物学组织奖学金(至C.C.);英国医学研究理事会和欧洲研究理事会(Advanced Grant 269058)授予M.d.B.

参考

  1. Chen,C.,Itakura,E.,Nelson,GM,Sheng,M.,Laurent,P.,Fenk,LA,Butcher,RA,Hegde,RS and de Bono,M。(2017)。 =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/28099418”target =“_ blank”> IL-17是秀丽隐杆线虫感觉的神经调节剂回复。 自然 542(7639):43-48。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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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