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Trypsin Sensitivity Assay to Study the Folding Status of Proteins
胰蛋白酶敏感性试验研究蛋白质的折叠状态   

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Abstract

This protocol aims to evaluate folding status of proteins, utilizing trypsin sensitivity. Unfolded/misfolded proteins are more susceptible to trypsin than folded proteins, because trypsin easily accesses and cleaves loosely folded parts of proteins. This method is especially useful to compare tightness of the folding among wild-type and mutant proteins. As trypsin generally cleaves a peptide bond at the carboxyl-terminal side of the amino acids lysine or arginine, this method can be used to analyze the folding status of different types of proteins such as integral membrane or soluble proteins (Ninagawa et al., 2015) and is applicable to cell lysates of any species and tissues as well as to recombinant proteins. You can use this technique with regular molecular and cell biology equipment.

Keywords: Trypsin(胰蛋白酶), Protein folding(蛋白质折叠), Integral membrane and soluble proteins(积分膜蛋白和可溶性)

Materials and Reagents

  1. 6 well dish (Corning, Falcon®, catalog number: 353046 )
  2. PVDF membrane (GE Healthcare, catalog number: 10600023 )
  3. DT40 cell line (DT40 is a B cell line derived from an avian leukosis virus induced bursal lymphoma in a white leghorn chicken) (ATCC, catalog number: CRL-2111 )
  4. Homo sapiens colon colorectal carcinoma cell line (HCT116) (ATCC, catalog number: CCL-247 )
  5. Dulbecco’s modified Eagle’s medium (DMEM) (NACALAI TESQUE, catalog number: 08458-45 )
  6. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  7. 100 U/ml penicillin and 100 μg/ml streptomycin (NACALAI TESQUE)
  8. RPMI (NACALAI TESQUE, catalog number: 30263-95 )
  9. Chicken serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16110-082 )
  10. Opti-mem (Thermo Fisher Scientific, GibcoTM, catalog number: 31985-070 )
  11. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  12. 2.5 g/L trypsin (NACALAI TESQUE, catalog number: 32777-44 )
  13. Protease inhibitor cocktail (100x) (NACALAI TESQUE, catalog number: 25955-11 ) (for inhibition of various proteases’ activity such as trypsin, in ddH2O; stored at -20 °C)
  14. NaCl (Wako Pure Chemical Industries, catalog number: 191-01665) (for DT40)
  15. Na2HPO4
  16. KCl
  17. KH2PO4
  18. Tris/HCl (pH 8.0) (Sigma-Aldrich, catalog number: T6791 ) (Stored at room temperature)
  19. Glycerol
  20. Bromophenol blue (BPB)
  21. Sodium dodecyl sulfate
  22. Nonidet P-40 (NACALAI TESQUE, catalog number: 23640-94 ) (for HCT116)
  23. 20 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) (Promega, catalog number: G7231 ) (for inhibition of PNGase activity)
  24. 10 mM Z-Leu-Leu-Leu-CHO (MG132) (PEPTIDE INSTITUTE, catalog number: 3175-v ) (in DMSO; inhibition of proteasomal activity, stored at -20 °C)
  25. 1 M dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 ) (in water; for reduction of proteins)
  26. Anti-β-actin antibody (Wako Pure Chemical Industries, catalog number: 017-24573 )
  27. Anti-myc antibody (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 562 )
  28. Phosphate buffered saline (PBS) (see Recipes)
  29. 2x sodium dodecyl sulfate (SDS) sample buffer (pH 6.8) (see Recipes)
  30. Buffer A (see Recipes)
  31. Buffer B (see Recipes)

Equipment

  1. High speed refrigerated micro centrifuge (Tomy, model: MX-301 )
  2. mPAGE (ATTO, model: AE-6530 ) (Using hand-made 10% gel)
  3. Transfer equipment (ATTO, model: WSE-4020 )
  4. Heat block (TAITEC, model: DTU-1BN )
  5. Micro porator (Digital Bio, model: MP-100 )

Software

  1. ImageJ

Procedure

  1. Culture adherent HCT116 cells in Dulbecco’s modified Eagle’s medium (glucose 4.5 g/L) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37 °C in a humidified 5% CO2/95% air atmosphere. Culture suspended DT40 cells at a density of 1 x 105-1 x 106 cells per ml in RPMI1640 medium supplemented with 10% fetal bovine serum, 1% chicken serum and antibiotics (100 U/ml penicillin and 100 ug/ml streptomycin) at 39.5 °C in a humidified 5% CO2/95% air atmosphere.
  2. Transfect 8.0 x 105 HCT116 cells (40-60% confluent in 6-well plate) using 1 μg of plasmid DNA, 300 μl of Opti-mem and 10 μl of Lipofectamine 2000 (Invitrogen) for one well according to the manufacturers’ instructions (After the transfection medium is not replaced). To obtain 1.05 x 107 cells transfected cells, electroporate three times 3.5 x 106 DT40 cells with 8 µg plasmid DNA with two pulses at 1,500 V for 15 msec according to the manufacturer’s instructions.
  3. Collect approximately 2.0 x 106 HCT116 cells or 4.0 x 106 DT40 cells for further analysis 24 h (HCT116 cell) or 16 h (DT40 cells) after transfection. For this the cells are washed with 1 ml PBS three times, scraped off and centrifuged at 3,000 x g for 2 min at 4 °C. Discard the supernatants of samples and suspend cell pellets in 400 μl (for HCT116 cells) or 250 µl (for DT40 cells) buffer A and incubate for 20 min on ice to lyse the cells. We used smaller amount of buffer A for DT40 cells, because DT40 cells do not contain as much proteins as HCT116 cells. After this step, samples from HCT116 cells and DT40 cells are treated in the same manner.
    Note: Important, the buffer A must NOT contain protease inhibitor cocktail as it would inhibit trypsin activity.
  4. Clarify cell lysates by centrifugation at 17,800 x g for 10 min at 4 °C and transfer the supernatants to new tubes. Pellets are discarded.
  5. To allow trypsin processing of misfolded proteins, incubate 50 µl aliquots (in case of protein concentrations of 1 mg/ml) of the cleared lysates with 1 µl trypsin solution (Test different concentrations from 1 µg/ml to 100 µg/ml) for 15 min at 4 °C. To stop the trypsin reaction, mix samples with 40-50 μl of buffer B supplemented with 10x protease inhibitor cocktail (We usually use high inhibitor concentrations: 5x final) and incubate at 100 °C for 5 min. Important, every 3 min four samples could be handled for equal incubation time with trypsin.
  6. Separate 10 µl of the samples (approximately 5 µg) by SDS-PAGE followed by immunoblotting using PVDF membrane and probing with an antibody against the protein of interest. Percentage of SDS-PAGE depends on the molecular weight of the protein of interest. Band intensities can be analyzed and compared to each other by ImageJ or similar programs (Figure 1).


    Figure 1. Severely misfolded mutants were more sensitive to trypsin. A. Chicken EDEM1/2/3 triple KO cells (gEDEM TKO) were described previously (Ninagawa et al., 2015). The indicated myc-tagged hATF6α(C) variants were transiently express in gEDEM-TKO cells and subjected to trypsin sensitivity assay. After the indicated time points the trypsin reaction was stopped and samples analyzed by a 12% SDS-PAGE followed by immunoblotting with antibodies against the myc-tag. The data shown represents a single representative experiment out of three repeats. Less folded proteins are more sensitive to trypsin, so the hATF6α(C)-myct mutants, Δ111-119, 182-194 mutant and Δ219-270 mutant were cleaved by lower trypsin concentrations. B. Quantification of band intensities shown in (A) by ImageJ and calculation of the percentage of remaining protein. C. Equal loading of cell lysates shown by immunoblotting of the lysates and probing with an antibody against β-actin.

Notes

  1. Incubation time with trypsin and concentration of trypsin should be optimized for proteins of interests.
  2. The lot and freshness of trypsin can affect the result and can change the outcome between independent experiments (We use trypsin stored at 4 °C). Anyway, you can compare the tightness of the folding among proteins of interests.
  3. Dilute trypsin in PBS to obtain the expected concentration.
  4. We used gEDEM TKO cells. This method is applicable for a wide variety of cells lines and recombinant proteins.
  5. This method can be applied to recombinant proteins. In this case, you might reduce concentration of trypsin.
  6. CPY and CPY* (Izawa et al., 2012), and GFP and GFP variants (Xu et al., 2013) were other examples used in this assay. We can provide plasmids to express hATF6α(C)-myct 1-302 and Δ280-298 for positive controls (Folded), and Δ111-119, 182-194 and Δ219-270 for negative controls (Less folded).

Recipes

  1. Phosphate buffered saline (PBS)
    137 mM NaCl
    8.1 mM Na2HPO4
    2.68 mM KCl
    1.47 mM KH2PO4
  2. 2x sodium dodecyl sulfate (SDS) sample buffer (pH 6.8)
    100 mM Tris/HCl (pH 6.8)
    20% glycerol
    0.2% bromophenol blue (BPB)
    4% sodium dodecyl sulfate
  3. Buffer A
    50 mM Tris/HCl (pH 8.0)
    1% NP-40
    150 mM NaCl
    2 μM Z-VAD-fmk (Add before use)
    20 μM MG132 (Add before use)
    Stored at 4 °C.
  4. Buffer B
    1x SDS sample buffer
    100 mM dithiothreitol (Add before use)
    10x protease inhibitor cocktail (Add before use, and 10 times diluted from original protease inhibitor cocktail)
    Stored at room temperature.

Acknowledgments

This protocol was adapted from and used in Ninagawa et al. (2015), Izawa et al. (2012) and Xu et al. (2013).

References

  1. Izawa, T., Nagai, H., Endo, T. and Nishikawa, S. (2012). Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation. Mol Biol Cell 23(7): 1283-1293.
  2. Ninagawa, S., Okada, T., Sumitomo, Y., Horimoto, S., Sugimoto, T., Ishikawa, T., Takeda, S., Yamamoto, T., Suzuki, T., Kamiya, Y., Kato, K. and Mori, K. (2015). Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway. J Cell Biol 211(4): 775-784.
  3. Xu, C., Wang, S., Thibault, G. and Ng, D. T. (2013). Futile protein folding cycles in the ER are terminated by the unfolded protein O-mannosylation pathway. Science 340(6135): 978-981.

简介

该协议旨在评估蛋白质的折叠状态,利用胰蛋白酶敏感性。 由于胰蛋白酶容易进入和切割松散折叠的蛋白质部分,展开的/错误折叠的蛋白质比折叠的蛋白质更易于胰蛋白酶。 这种方法特别适用于比较野生型和突变型蛋白质之间折叠的紧密度。 由于胰蛋白酶通常在氨基酸赖氨酸或精氨酸的羧基末端侧切割肽键,所以该方法可用于分析不同类型蛋白质如整合膜或可溶性蛋白质的折叠状态(Ninagawa等,2015 ),适用于任何物种和组织以及重组蛋白的细胞裂解物。 您可以使用这种技术与常规分子和细胞生物学设备。

关键字:胰蛋白酶, 蛋白质折叠, 积分膜蛋白和可溶性

材料和试剂

  1. 6孔皿(Corning,Falcon ,目录号:353046)
  2. PVDF膜(GE Healthcare,目录号:10600023)
  3. DT40细胞系(DT40是来源于白喉毒鸡中的禽白血病病毒诱导的淋巴管淋巴瘤的B细胞系)(ATCC,目录号:CRL-2111)
  4. 智人结肠结肠直肠癌细胞系(HCT116)(ATCC,目录号:CCL-247)
  5. Dulbecco改良的Eagle培养基(DMEM)(NACALAI TESQUE,目录号:08458-45)
  6. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
  7. 100U/ml青霉素和100μg/ml链霉素(NACALAI TESQUE)
  8. RPMI(NACALAI TESQUE,目录号:30263-95)
  9. 鸡血清(Thermo Fisher Scientific,Gibco TM ,目录号:16110-082)
  10. Opti-mem(Thermo Fisher Scientific,Gibco TM ,目录号:31985-070)
  11. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM ,目录号:11668019)
  12. 2.5g/L胰蛋白酶(NACALAI TESQUE,目录号:32777-44)
  13. 蛋白酶抑制剂混合物(100x)(NACALAI TESQUE,目录号:25955-11)(用于抑制各种蛋白酶活性,例如在ddH 2 O中的胰蛋白酶;储存在-20℃) />
  14. NaCl(Wako Pure Chemical Industries,目录号:191-01665)(用于DT40)
  15. Na HPO 4
  16. KCl
  17. KH 2 PO 4
  18. Tris/HCl(pH8.0)(Sigma-Aldrich,目录号:T6791)(在室温下保存)
  19. 甘油
  20. 溴酚蓝(BPB)
  21. 十二烷基硫酸钠
  22. Nonidet P-40(NACALAI TESQUE,目录号:23640-94)(用于HCT116)
  23. 20mM苄氧羰基 - 缬氨酰 - 丙氨酰 - 天冬氨酰基 - [O-甲基] - 氟甲基酮(Z-VAD-fmk)(Promega,目录号:G7231)(用于抑制PNGase活性)
  24. 10mM Z-Leu-Leu-Leu-CHO(MG132)(PEPTIDE INSTITUTE,目录号:3175-v)(DMSO中;抑制蛋白酶体活性,-20℃贮存)
  25. 将1M二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:041-08976)(在水中;用于蛋白质还原)
  26. 抗β-肌动蛋白抗体(Wako Pure Chemical Industries,目录号:017-24573)
  27. 抗myc抗体(MEDICAL& BIOLOGICAL LABORATORIES,目录号:562)
  28. 磷酸盐缓冲盐水(PBS)(见Recipes)
  29. 2x十二烷基硫酸钠(SDS)样品缓冲液(pH 6.8)(参见配方)
  30. 缓冲区A(参见配方)
  31. 缓冲液B(参见配方)

设备

  1. 高速冷藏微量离心机(Tomy,型号:MX-301)
  2. mPAGE(ATTO,型号:AE-6530)(使用手工制备的10%凝胶)
  3. 传输设备(ATTO,型号:WSE-4020)
  4. 加热块(TAITEC,型号:DTU-1BN)
  5. 微孔器(Digital Bio,型号:MP-100)

软件

  1. ImageJ

程序

  1. 在37℃,潮湿的5%CO 2中,将补充有10%胎牛血清和抗生素(100U/ml青霉素和100μg/ml链霉素)的Dulbecco改良的Eagle培养基(葡萄糖4.5g/L)中的贴壁HCT116细胞培养> 2/95%空气气氛。培养物在补充有10%胎牛血清,1%鸡血清的RPMI1640培养基中以1×10 5 -1×10 6个细胞/ml的密度悬浮DT40细胞,抗生素(100U/ml青霉素和100ug/ml链霉素)中在39.5℃下在潮湿的5%CO 2/95%空气气氛中培养。
  2. 使用1μg质粒DNA,300μlOpti-mem和10μlLipofectamine 2000(Invitrogen),转染8.0×10 5个HCT116细胞(在6孔板中40-60%汇合)根据厂家说明(转染后培养基不更换)。为了获得1.05×10 7个细胞转染的细胞,用8μg质粒DNA电穿孔3次3.5×10 6个DT40细胞,两次脉冲1500V,15msec,根据制造商的说明
  3. 在转染后收集约2.0×10 6个HCT116细胞或4.0×10 6个DT40细胞用于进一步分析24小时(HCT116细胞)或16小时(DT40细胞)。为此,用1ml PBS洗涤细胞三次,刮下并在4℃以3,000xg离心2分钟。弃去样品的上清液,悬浮细胞沉淀在400微升(对于HCT116细胞)或250微升(对于DT40细胞)缓冲液A中,在冰上孵育20分钟以裂解细胞。我们使用较小量的缓冲液A用于DT40细胞,因为DT40细胞不含有与HCT116细胞一样多的蛋白质。在该步骤之后,以相同的方式处理来自HCT116细胞和DT40细胞的样品 注意:重要的是,缓冲液A不能含有蛋白酶抑制剂混合物,因为它会抑制胰蛋白酶活性。
  4. 通过在4℃下以17,800×g离心10分钟澄清细胞裂解物,并将上清液转移到新管中。弃去颗粒。
  5. 为了允许胰蛋白酶处理错误折叠的蛋白质,用1μl胰蛋白酶溶液(测试不同浓度从1μg/ml至100μg/ml)将50μl等分试样(在蛋白质浓度为1mg/ml的情况下)孵育15分钟min。为了停止胰蛋白酶反应,将样品与40-50μl补充有10x蛋白酶抑制剂混合物(我们通常使用高抑制剂浓度:5x最终)的缓冲液B混合,并在100℃孵育5分钟。重要的是,每3分钟4个样品可以处理与胰蛋白酶相等的孵育时间
  6. 通过SDS-PAGE分离10μl样品(约5μg),然后使用PVDF膜进行免疫印迹,并用针对目的蛋白的抗体进行探测。 SDS-PAGE的百分比取决于目标蛋白质的分子量。可以通过ImageJ或类似程序(图1)分析和比较谱带强度

    图1.严重错折叠突变体对胰蛋白酶更敏感。A.先前描述了鸡EDEM1/2/3三重KO细胞(gEDEM TKO)(Ninagawa等人, 2015)。指示的myc标记的hATF6α(C)变体在gEDEM-TKO细胞中瞬时表达,并进行胰蛋白酶敏感性测定。在指定的时间点后,停止胰蛋白酶反应并通过12%SDS-PAGE分析样品,然后用针对myc标签的抗体进行免疫印迹。所示数据代表三个重复中的单个代表性实验。较少折叠的蛋白质对胰蛋白酶更敏感,因此hATF6α(C)-myct突变体Δ111-119,182-194突变体和Δ219-270突变体被较低的胰蛋白酶浓度切割。 B.通过ImageJ显示(A)中的条带强度的定量并计算剩余蛋白质的百分比。 C.等量装载细胞裂解物,通过裂解物的免疫印迹显示并用抗β-肌动蛋白的抗体探测。

笔记

  1. 与胰蛋白酶的孵育时间和胰蛋白酶的浓度应针对感兴趣的蛋白质进行优化。
  2. 胰蛋白酶的批次和新鲜度可以影响结果并且可以改变独立实验之间的结果(我们使用在4℃下储存的胰蛋白酶)。无论如何,你可以比较蛋白质之间的折叠的紧张感兴趣。
  3. 在PBS中稀释胰蛋白酶以获得预期浓度
  4. 我们使用gEDEM TKO细胞。该方法适用于多种细胞系和重组蛋白。
  5. 该方法可应用于重组蛋白。在这种情况下,您可能会降低胰蛋白酶的浓度。
  6. CPY和CPY *(Izawa等人,2012),以及GFP和GFP变体(Xu等人,2013)是本测定中使用的其他实例。我们可以提供质粒来表达用于阳性对照(折叠)的hATF6α(C)-myct 1-302和Δ280-298,以及用于阴性对照(折叠较少)的Δ111-119,182-194和Δ219-270。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl 8.1mM Na 2 HPO 4
    2.68mM KCl
    1.47mM KH 2 PO 4 sub/
  2. 2x十二烷基硫酸钠(SDS)样品缓冲液(pH 6.8) 100mM Tris/HCl(pH6.8)
    20%甘油 0.2%溴酚蓝(BPB)
    4%十二烷基硫酸钠
  3. 缓冲区A
    50mM Tris/HCl(pH8.0) 1%NP-40
    150mM NaCl 2μMZ-VAD-fmk(使用前添加)
    20μMMG132(使用前添加)
    储存在4°C。
  4. 缓冲区B
    1x SDS样品缓冲液
    100 mM二硫苏糖醇(使用前加入)
    10x蛋白酶抑制剂混合物(使用前加入,从原始蛋白酶抑制剂混合物稀释10倍) 室温保存。

致谢

该协议改编自Ninagawa等人(2015),Izawa等人(2012)和Xu等人的 。 (2013年)。

参考文献

  1. Izawa,T.,Nagai,H.,Endo,T.and Nishikawa,S.(2012)。  Yos9p和Hrd1p介导错误折叠的蛋白质对ER相关降解的ER保留。 Mol Biol Cell 23(7):1283-1293。
  2. Ninagawa,S.,Okada,T.,Sumitomo,Y.,Horimoto,S.,Sugimoto,T.,Ishikawa,T.,Takeda,S.,Yamamoto,T.,Suzuki,T.,Kamiya, Kato,K. and Mori,K.(2015)。  通过非糖蛋白ERAD途径强制破坏严重错误折叠的哺乳动物糖蛋白。细胞生物学211(4):775-784。
  3. Xu,C.,Wang,S.,Thibault,G.and Ng,DT(2013)。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ninagawa, S. and Mori, K. (2016). Trypsin Sensitivity Assay to Study the Folding Status of Proteins. Bio-protocol 6(19): e1953. DOI: 10.21769/BioProtoc.1953.
  2. Ninagawa, S., Okada, T., Sumitomo, Y., Horimoto, S., Sugimoto, T., Ishikawa, T., Takeda, S., Yamamoto, T., Suzuki, T., Kamiya, Y., Kato, K. and Mori, K. (2015). Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway. J Cell Biol 211(4): 775-784.
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