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In vitro Chitin Binding Assay
体外几丁质结合试验   

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参见作者原研究论文

本实验方案简略版
Journal of Bacteriology
Jul 2014

Abstract

Chitin is polymer of N-acetylglucosamine (GlcNAc) found in the exoskeleton of arthropods and the fungal cell wall. GlcNAc is also implicated in bacterial development, adherence, and signal transduction but can also be used as a carbon source. In vitro chitin binding assay is performed to determine the affinity of a purified protein to the chitin molecule. The principle is based on the co-sedimentation of chitin-binding proteins together with chitin-coated beads.

Keywords: Chitin (甲壳素), N acetylglucosamine (N-乙酰氨基葡萄糖), Chitin beads (Chitin珠子), Co-purification (CO净化), Affinity (密切关系)

Materials and Reagents

  1. Purified protein with chitin binding affinity
    Note: We used histidine-tagged chitin binding protein CbpD. The protein was purified by affinity chromatography onto a 5-ml HisTrap nickel column (Pharmacia) on an Äkta system (Amersham Biosciences). The complete purification protocol is described in details in Cadoret et al. (2014).
  2. Chitin beads (New England Biolabs, catalog number: S6651 )
  3. Tris Base
  4. EDTA
  5. NaCl
  6. Tween 100
  7. Freshly made solution of chitin binding buffer (see Recipes)

Equipment

  1. Laboratory vortex adapted to Eppendorf tubes
  2. Laboratory rotating wheel adapted to Eppendorf tubes
  3. Cold room or refrigerated incubator (4 °C)

Procedure

  1. Wash the chitin beads as follows
    1. Vortex the chitin beads before pipetting.
    2. Wash the desired amount of beads solution (100 µl per experiment) twice with 5 volumes of chitin binding buffer. This washing step is performed by gravity flow by leaving the sample 5 min at room temperature.

  2. Binding assay
    1. Prepare a 200 µl solution of chitin-binding buffer containing the purified CbpD protein at 60 µg/ml (total fraction). For that, 1.5 µl of purified CbpD at 8 mg/ml in 50 mM Tris pH 8, 300 mM NaCl, 250 mM imidazole was mixed with 198. 5 µl of chitin binding buffer.
    2. Add 100 µl of pre-washed chitin beads in chitin-binding buffer to the purified protein solution.
    3. Incubate the sample on a laboratory rotating wheel for 90 min at 4 °C.
    4. After gravity flow separation, collect the supernatant (unbound fraction).
    5. Wash the beads (bound fraction) twice with 200 µl of chitin binding buffer by gravity flow.
    6. Analyse 15 μl of Total (equivalent to 0.9 μg), Unbound and Bound fractions by SDS-PAGE followed by Coomassie blue staining and/or immunoblotting (see Figure 1).

Representative data



Figure 1. Chitin-binding affinity of CbpD. To test the chitin-binding affinity of CbpD, a solution of purified CbpD at 200 μg/ml in chitin binding buffer (Total) was incubated with chitin beads according to the present protocol. 15 μl samples of Total, Unbound and Bound fractions were analyzed by SDS-PAGE followed by Coomassie blue staining. Molecular mass markers (in kDa) are indicated on the left.

Notes

  1. The chitin binding buffer and the protein solution in chitin binding buffer must be freshly prepared.
  2. A negative control can consist of the same experiment where the chitin beads solution is replaced by chitin binding buffer.
  3. Additionally, purified proteins without chitin binding affinity such as Elastase (LasB) and exotoxin A (ToxA) can be used as negative control [see Figure 5B in Cadoret et al. (2014)].

Recipes

  1. Freshly made solution of chitin binding buffer
    Chitin binding buffer:
    50 mM Tris HCl (pH 8)
    1 mM EDTA (pH 8)
    500 mM NaCl
    0.1% Tween 100
    The chitin binding buffer is prepared from stock solutions:
    Tris HCl 1 M (pH 8)
    EDTA 0.5 M (pH 8)
    NaCl 5 M

Acknowledgments

This protocol adapted from Shutinoski et al. (2010) was used in Cadoret et al. (2014). The work is funded by a Ph.D. grant from the French government.

References

  1. Cadoret, F., Ball, G., Douzi, B. and Voulhoux, R. (2014). Txc, a new type II secretion system of Pseudomonas aeruginosa strain PA7, is regulated by the TtsS/TtsR two-component system and directs specific secretion of the CbpE chitin-binding protein. J Bacteriol 196(13): 2376-2386.
  2. Shutinoski, B., Schmidt, M. A. and Heusipp, G. (2010). Transcriptional regulation of the Yts1 type II secretion system of Yersinia enterocolitica and identification of secretion substrates. Mol Microbiol 75(3): 676-691.

简介

几丁质是在节肢动物和真菌细胞壁的外骨骼中发现的N-乙酰葡萄糖胺(GlcNAc)的聚合物。 GlcNAc还涉及细菌发育,粘附和信号转导,但也可以用作碳源。 进行体外几丁质结合测定以测定纯化的蛋白质与几丁质分子的亲和力。 该原理基于几丁质结合蛋白与壳多糖包被的珠的共沉淀。

关键字:甲壳素, N-乙酰氨基葡萄糖, Chitin珠子, CO净化, 密切关系

材料和试剂

  1. 具有几丁质结合亲和力的纯化蛋白质 注意:我们使用组氨酸标记的几丁质结合蛋白CbpD。 通过亲和层析将蛋白质在Äkta系统(Amersham Biosciences)上的5ml HisTrap镍柱(Pharmacia)上纯化。 完全纯化方案详细描述于Cadoret等人 (2014)。
  2. 壳多糖珠(New England Biolabs,目录号:S6651)
  3. Tris碱
  4. EDTA
  5. NaCl
  6. 吐温100
  7. 新鲜制备的几丁质结合缓冲液溶液(见配方)

设备

  1. 实验室涡流适应于Eppendorf管
  2. 实验室旋转轮适用于Eppendorf管
  3. 冷室或冷藏培养箱(4℃)

程序

  1. 按照以下步骤清洗几丁质珠子
    1. 移液前涡旋几丁质珠。
    2. 洗涤所需的 珠量溶液(每次实验100μl),用5体积的量两次   几丁质结合缓冲液。 该洗涤步骤通过重力流进行 通过将样品在室温下放置5分钟

  2. 结合测定
    1. 准备一个200微升几丁质结合缓冲液的溶液,含有 纯化的CbpD蛋白(60μg/ml)(总分数)。 为此,1.5微升 纯化的CbpD,8mg/ml,在50mM Tris pH 8,300mM NaCl,250mM中 咪唑与198.8μl几丁质结合缓冲液混合。
    2. 加入100微升预洗涤几丁质珠在几丁质结合缓冲液到纯化的蛋白质溶液
    3. 将样品在实验室旋转轮上在4℃孵育90分钟
    4. 重力流分离后,收集上清液(未结合部分)
    5. 用200μl甲壳质结合缓冲液通过重力流冲洗珠子(结合级分)两次
    6. 分析总共15μl(相当于0.9μg),未结合和结合 通过SDS-PAGE,随后考马斯蓝染色和/或 免疫印迹(参见图1)。

代表数据



图1。 CbpD的几丁质结合亲和力。为了测试CbpD的几丁质结合亲和力,将200μg/ml的纯化CbpD在几丁质结合缓冲液)与根据本方案的几丁质珠孵育。通过SDS-PAGE,然后考马斯蓝染色分析总共,未结合和结合级分的15μl样品。分子量标记(以kDa表示)在左侧显示。

笔记

  1. 几丁质结合缓冲液和几丁质结合缓冲液中的蛋白质溶液必须新鲜制备
  2. 阴性对照可以由相同的实验组成,其中几丁质珠溶液被几丁质结合缓冲液替代。
  3. 此外,没有壳多糖结合亲和力的纯化蛋白如弹性蛋白酶(LasB)和外毒素A(ToxA)可用作阴性对照[参见Cadoret等人(2014)中的图5B]。br/>

食谱

  1. 新鲜制备的几丁质结合缓冲液溶液
    几丁质结合缓冲液:
    50mM Tris HCl(pH8)
    1mM EDTA(pH8)
    500 mM NaCl
    0.1%Tween 100
    几丁质结合缓冲液由储备溶液制备:
    Tris HCl 1M(pH 8)
    EDTA 0.5M(pH8)
    NaCl 5×m/v

致谢

在Cadoret等人(2014)中使用从Shutinoski等人(2010)改编的该协议。 这项工作由博士学位资助。 来自法国政府的赠款。

参考文献

  1. Cadoret,F.,Ball,G.,Douzi,B。和Voulhoux,R。(2014)。 Txc,一种新型的绿脓杆菌菌株PA7的II型分泌系统, 受TtsS/TtsR双组分系统调节并指导CbpE几丁质结合蛋白的特异性分泌。细菌学杂志196(13):2376-2386。
  2. Shutinoski,B.,Schmidt,M.A。和Heusipp,G。(2010)。 Yersinia enterocolitica的Yts1 II型分泌系统的转录调控和鉴定 的分泌底物。 Mol Microbiol 75(3):676-691。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ball, G., Cadoret, F. and Voulhoux, R. (2015). In vitro Chitin Binding Assay. Bio-protocol 5(4): e1401. DOI: 10.21769/BioProtoc.1401.
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