搜索

Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans
构巢曲霉中表达的GfsA-3xFLAG 蛋白的纯化   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces cerevisiae (S. cerevisiae). Therefore, we decided to employ Aspergillus nidulans (A. nidulans) as the host organism, and produced a strain that expressed 3x FLAG-tagged GfsA using chromosomal tagging. To confirm its expression, a solubilized protein was prepared from the tagged strain and analyzed with an anti-FLAG antibody. The strain that expressed 3x FLAG-tagged GfsA produced a functional protein with a mass of approximately 67 kDa. The method described in this manuscript allows purification of the GfsA-3xFLAG protein as expressed in A. nidulans cells.

Keywords: Galactofuranose(呋喃), Galactofuranosyltransferase(galactofuranosyltransferase), Aspergillus(曲霉), Protein purification(蛋白纯化)

Materials and Reagents

  1. Aspergillus nidulans expressing 3x FLAG-tagged GfsA (Komachi et al., 2013)
  2. 3x FLAG-peptide (Sigma-Aldrich, catalog number: F4799 )
  3. ANTI-FLAG M2-agarose produced from mouse (Sigma-Aldrich, catalog number: A2220 )
  4. Mouse IgG-agarose (Sigma-Aldrich, catalog number: A0919 )
  5. 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) (Dojindo Molecular Technologies, catalog number: GB10 )
  6. Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
  7. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  8. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
  9. Manganese (II) chloride tetrahydrate (MnCl2) (Wako Pure Chemical Industries, catalog number: 133-00725 )
  10. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00611 )
  11. 3-[(3-Cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO) (Dojindo Molecular Technologies, catalog number: C020 )
  12. CompleteTM protease inhibitor cocktail tablets (EDTA-free) (Roche Diagnostics, catalog number: 1873580 )
  13. Liquid nitrogen
  14. Buffer A (see Recipes)
  15. Minimal medium (MM) (see Recipes)
  16. Hutner's trace elements (see Recipes)

Equipment

  1. Spreader
  2. 500-ml Sakaguchi flasks
  3. Mortar and pestle
  4. Aspirator
  5. Centrifuge with an angle rotor
  6. Centrifuge with a swing rotor
  7. Ultracentrifuge
  8. Spatula
  9. 15-ml plastic centrifuge tube (e.g., Greiner Bio-One GmbH)
  10. 4 °C incubator
  11. 30 °C incubator
  12. Rotator (e.g., TAITEC)
  13. Filter paper (Munktell & Filtrak GmbH, catalog number: 113053 )

Procedure

  1. Streak Aspergillus nidulans conidia from frozen stock onto Minimal medium (MM) plate and cultivate for 3 days at 30 °C.
  2. Collect the formed conidia with a spreader.
  3. Spread Aspergillus nidulans conidia (1 x 105) onto MM plates and cultivate for 3 days at 30 °C.
  4. Inoculate the collected conidia (2 x 107) into 100 ml of MM in 500-ml Sakaguchi flasks.
  5. Shake the flasks at 126 rpm at 30 °C for 24 h.
  6. Collect the mycelial cells by paper filtration.
  7. Wash the cells twice with approximately 30 ml of distilled water.
    Note: Cells can easily be crushed by wringing wet cells out to dry using a scoopula after this step as much as possible.
  8. Grind cells (25 g of wet cells) into a fine powder in liquid nitrogen using a mortar and pestle.
  9. Resuspend the lysed cells in 100 ml of buffer A containing CompleteTM protease inhibitor cocktail (EDTA-free).
  10. Remove cell debris by centrifugation with an angle rotor at 10,000 x g for 10 min.
  11. Centrifuge the supernatant at 100,000 x g for 45 min using an ultracentrifuge.
  12. Resuspend the resultant pellet in 10 ml of buffer A containing 0.5% CHAPSO using a spatula.
    Note: Pilot experiments are needed to determine the suitable conditions under which the detergents solubilize the target protein.
  13. Gently mix the sample for 1 h using a rotator to obtain solubilized membrane proteins.
  14. Centrifuge the sample at 100,000 x g for 30 min using an ultracentrifuge.
  15. Collect the supernatant (approximately 10 ml) into a 15-ml plastic centrifuge tube.
  16. Add mouse-IgG-agarose (100 µl) to the supernatant and gently shake the mixture for 1 h (Video 1).

    Video 1. Shaking the mixture using a rotator

  17. Remove the mouse-IgG-agarose by centrifugation with a swing rotor at 1,400 x g for 10 min.
  18. Add 200 µl anti-FLAG M2 affinity gel to the supernatant and gently shake the resultant mixture for 1 h.
  19. Collect the Anti-FLAG M2 affinity gel by centrifugation with a swing rotor at 1,400 x g for 10 min.
  20. Gently remove the supernatant with an aspirator.
  21. Resuspend the resultant agarose with 15 ml of buffer A containing 0.1% CHAPSO.
  22. Repeat steps 18-20 five times.
  23. Elute GfsA protein with 20 µl of buffer A with 0.1% CHAPSO containing 0.5 µg/µl 3x FLAG peptide.


    Figure 1. Purification of GfsA-3xFLAG protein. A total of 0.5 mg (silver staining) proteins were separated by 5%-20% SDS-PAGE, and were then assayed by silver staining. GfsA-3xFLAG was detected as a 67 kDa protein. Asterisk indicates a degradation product or insufficiently N-glycosylated product of GfsA-3xFLAG.

Notes

  1. Perform all manipulations on ice or at 4 °C.

Recipes

  1. Buffer A (1 L)
    Compounds
    Amounts
    HEPES            
    11.9 g
    NaCl
    5.84 g
    KCl                   
    2.24 g
    MnCl2.4H2O
    0.2 g
    Glycerol
    50 g
    Add water to bring the final solution to 1 L total volume
    Filter sterilize the solution using a 0.45 μm filter
    Stored at 4 °C
  2. Minimal medium (1 L)
    Compounds
    Amounts
    NaNO                   
    6.0 g
    KCl
    0.52 g
    MgSO4.7H20
    0.52 g
    KH2PO4                 
    1.52 g
    Glucose
    10.0 g
    Hutner's trace elements
    2 ml
    Adjust pH to 6.8 using NaOH
    Add water to bring the final solution to 1 L total volume
    Autoclave for 20 min
  3. Hutner's trace elements
    Compounds
    Amounts
    H2O (60 °C)
    100 ml
    ZnSO4.7H2O
    2.2 g
    H3BO3  
    1.1 g
    MnCl2.4H2O
    0.5 g
    FeSO4.7H2O
    0.5 g
    CoCl2.6H2O                
    0.16 g
    CuSO4.5H2O
    0.16 g
    (NH4) 6Mo7O24.4H2O
    0.11 g
    EDTA 
    5.0  g
    Adjust the pH value to 6.5-6.8 using KOH

Acknowledgments

This protocol was adapted from the previously published paper Komachi et al., 2013. The work was supported in part by Grants-in-Aid for Young Scientists (B) from the Japan Society of the Promotion of Science (JSPS) (21780313, 23780350 and 26450106) (to T.O.).

References

  1. Komachi, Y., Hatakeyama, S., Motomatsu, H., Futagami, T., Kizjakina, K., Sobrado, P., Ekino, K., Takegawa, K., Goto, M., Nomura, Y. and Oka, T. (2013). GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol Microbiol 90(5): 1054-1073.

简介

GfsA是涉及O - 聚糖的生物合成的真菌β-半乳糖呋喃糖基转移酶。 为了研究GfsA的酶功能,我们尝试从两种异源宿主生物体获得该酶的重组蛋白。 然而,GfsA不能在大肠杆菌(大肠杆菌)或酿酒酵母(Saccharomyces cerevisiae)中表达为重组蛋白质( cerevisiae )。 因此,我们决定使用构巢曲霉( A。nidulans )作为宿主生物体,并产生使用染色体标签表达3×FLAG标记的GfsA的菌株。 为了证实其表达,从标记的菌株制备溶解的蛋白并用抗FLAG抗体分析。 表达3×FLAG标记的GfsA的菌株产生质量为约67kDa的功能性蛋白质。 该手稿中描述的方法允许纯化在A中表达的GfsA-3xFLAG蛋白。 构巢细胞。

关键字:呋喃, galactofuranosyltransferase, 曲霉, 蛋白纯化

材料和试剂

  1. 表达3x FLAG标记的GfsA(Komachi等人,2013),
  2. 3x FLAG-肽(Sigma-Aldrich,目录号:F4799)
  3. 从小鼠(Sigma-Aldrich,目录号:A2220)产生的抗FLAG M2-琼脂糖
  4. 小鼠IgG-琼脂糖(Sigma-Aldrich,目录号:A0919)
  5. 2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(HEPES)(Dojindo Molecular Technologies,目录号:GB10)
  6. 氢氧化钠(NaOH)(Wako Pure Chemical Industries,目录号:198-13765)
  7. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  8. 氯化钾(KCl)(Wako Pure Chemical Industries,目录号:163-03545)
  9. 氯化锰(II)四水合物(MnCl 2)(Wako Pure Chemical Industries,目录号:133-00725)
  10. 甘油(Wako Pure Chemical Industries,目录号:075-00611)
  11. 3 - [(3-胆酰氨基丙基)二甲基铵基] -2-羟基丙磺酸盐(CHAPSO)(Dojindo Molecular Technologies,目录号:C020)
  12. 完全蛋白酶抑制剂混合物片剂(无EDTA)(Roche Diagnostics,目录号:1873580)
  13. 液氮
  14. 缓冲区A(参见配方)
  15. 最小介质(MM)(参见配方)
  16. Hutner的微量元素(参见配方)

设备

  1. 撒布机
  2. 500 ml Sakaguchi烧瓶
  3. 砂浆和杵
  4. 吸气器
  5. 用角转子离心
  6. 用旋转式搅拌器离心
  7. 超速离心机
  8. 小铲
  9. 15-ml塑料离心管(例如Greiner Bio-One GmbH)
  10. 4℃孵育器
  11. 30℃培养箱
  12. 旋转器(例如,TAITEC)
  13. 滤纸(Munktell& Filtrak GmbH,目录号:113053)

程序

  1. 将来自冷冻储存物的构巢曲霉分生孢子分生孢子在最小培养基(MM)平板上培养,并在30℃下培养3天。
  2. 使用撒布机收集形成的分生孢子。
  3. 将分生孢子(1×10 5)铺在MM平板上,在30℃培养3天。
  4. 将收集的分生孢子(2×10 7个)接种到500ml坂口烧瓶中的100ml MM中。
  5. 将烧瓶以126rpm在30℃摇动24小时
  6. 通过纸过滤收集菌丝体细胞
  7. 用约30ml蒸馏水洗涤细胞两次。
    注意:在这一步骤之后,尽可能使用勺子将湿细胞拧干,使细胞很容易被压碎。
  8. 使用研钵和研杵在液氮中将细胞(25g湿细胞)粉碎成细粉末。
  9. 将裂解的细胞重悬于100ml含有Complete TM蛋白酶抑制剂混合物(不含EDTA)的缓冲液A中。
  10. 通过用角转子以10,000×g离心10分钟除去细胞碎片。
  11. 使用超速离心机将上清液以100,000×g离心45分钟
  12. 使用刮刀将所得沉淀重悬于10ml含有0.5%CHAPSO的缓冲液A中。
    注意:需要进行中试实验以确定洗涤剂溶解目标蛋白的合适条件。
  13. 使用旋转器轻轻混合样品1小时,得到溶解的膜蛋白
  14. 使用超速离心机将样品以100,000×g离心30分钟
  15. 收集上清液(约10毫升)到15毫升塑料离心管中
  16. 加入小鼠IgG琼脂糖(100微升)到上清液,轻轻摇动混合物1小时(视频1)。

    视频1.使用旋转器振动混合物
                                           <! - [if!IE]> - > <! - <![endif] - >                                            

    要播放视频,您需要安装较新版本的Adobe Flash Player。

    获取Adobe Flash Player

    <! - [if!IE]> - >
    <! - <![endif] - >

  17. 通过用摆动转子以1,400×g离心10分钟除去小鼠IgG-琼脂糖。
  18. 向上清液中加入200μl抗FLAG M2亲和凝胶,轻轻摇动所得混合物1小时
  19. 通过用摆动转子在1,400×g下离心10分钟收集抗FLAG M2亲和凝胶。
  20. 用吸气器轻轻取出上清液。
  21. 用15ml含有0.1%CHAPSO的缓冲液A重悬所得的琼脂糖
  22. 重复步骤18-20五次。
  23. 用20μl含0.1%CHAPSO的缓冲液A(含0.5μg/μl3×FLAG肽)洗脱GfsA蛋白。


    图1. GfsA-3xFLAG蛋白的纯化通过5%-20%SDS-PAGE分离总共0.5mg(银染色)蛋白,然后通过银染法进行测定。 检测到GfsA-3xFLAG为67kDa蛋白。 星号表示GfsA-3xFLAG的降解产物或不足的N - 糖基化产物。

笔记

  1. 在冰上或在4°C进行所有操作。

食谱

  1. 缓冲液A(1L)
    化合物
    金额
    HEPES             
    11.9克
    NaCl
    5.84克
    KCl                   
    2.24克
    MnCl 2 4H O
    0.2 g
    甘油
    50克
    加水,使最终溶液的体积为1 L 使用0.45μm过滤器过滤灭菌溶液
    储存在4°C
  2. 最小培养基(1L)
    化合物
    金额
    NaNO 3                   
    6.0克
    KCl
    0.52克
    MgSO 4 7H <2> 0
    0.52克
    KH 2 PO 4                
    1.52克
    葡萄糖
    10.0克
    Hutner的微量元素
    2 ml
    用NaOH
    调节pH至6.8 加水,使最终溶液的体积为1 L 高压灭菌20分钟
  3. Hutner的微量元素
    化合物
    金额
    H 2 O(60℃)
    100 ml
    ZnSO 4 7H <2> 2.2 g
    H 3 BO 3  
    1.1克
    MnCl 2 4H 2 O
    0.5克
    FeSO 4 7H <2> O
    0.5克
    CoCl <2> 6H 2 O              
    0.16 g
    CuSO 4 5H sub 2 O
    0.16 g
    (NH 4)6 Mo 7+ O 24+。在本发明的一个实施方案中, O
    0.11克
    EDTA 
    5.0  g
    用KOH
    调节pH值至6.5-6.8

致谢

该协议改编自以前发表的论文Komachi等人,2013年。该工作部分由来自日本科学促进会的青年科学家的助学金(B)支持 (JSPS) (21780313,23780350和26450106)(至T.O.)。

参考文献

  1. Komachi,Y.,Hatakeyama,S.,Motomatsu,H.,Futagami,T.,Kizjakina,K.,Sobrado,P.,Ekino,K.,Takegawa,K.,Goto,M.,Nomura, Oka,T。(2013)。 GfsA 编码参与半乳糖呋喃糖抗原生物合成的新型半乳糖呋喃糖基转移酶 > 90 90(5):1054-1073 。
  • English
  • 中文翻译
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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Oka, T., Katafuchi, Y., Fukuda, K., Ekino, K., Goto, M. and Nomura, Y. (2014). Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans. Bio-protocol 4(17): e1222. DOI: 10.21769/BioProtoc.1222.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。