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Native BAD-1 Binding to Heparin-agarose
天然BAD-1与肝素琼脂糖的结合   

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Abstract

BAD-1 is an adhesin created by the dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis. We have determined that it has an affinity for heparin, which may explain its impact on virulence and human immune function as a number of cells related to immune function have heparin like moieties on their surfaces. This assay allows a quantification of binding between soluble BAD-1 and immobilized heparin.

Materials and Reagents

  1. Heparin-agarose resin (Sigma-Aldrich, catalog number: H6508 ) (prior to use it is washed 3x in five volumes of tricine buffer to eliminate free heparin)
  2. 10 μg of BAD-1 [purified according to the method of Brandhorst et al. (2005)]
  3. 25 microliters of soluble medical-grade sodium heparin for injection (50 mg/ml) (Elkins-Sinn Inc)
  4. Tricine buffer (Sigma-Aldrich, catalog number: T0377 ) (see Recipes)

Equipment

  1. Accuspin micro17 microcentrifuge (Thermo Fisher Scientific)
  2. Nanodrop ND1000 spectrophotometer

Procedure

  1. 100 μl of 0.1 mg/ml BAD-1 in tricine buffer was incubated with agarose heparin resin (5 μl bed volume) for 30 min at 25 °C with occasional agitation.
  2. Resin beads were pelleted by centrifugation in an Accuspin microfuge at 7,000 x g for 1 min.
  3. Samples of the BAD-1 containing supernatant were analyzed for optical density at 280 nm in a Nanodrop ND1000 spectrophotometer. This reading was compared to a control solution of BAD-1 to which tricine buffer was added in place of heparin-agarose beads. The discrepancy in absorbance is assumed to be linear with respect to BAD-1 adhering to the heparin resin bed.
  4. Binding inhibition studies were done by repeating steps 1-3 in the presence of soluble medical grade heparin in tricine buffer. Heparin was added to the tricine binding buffer at various concentrations (0.01, 0.1 and 1 mg/ml - significant inhibition was noted at 0.1 mg/ml heparin and above.). Measurements of baseline absorbance were corrected to account for absorbance of added heparin inhibitor.

Recipes

  1. Tricine buffer
    20 mM tricine (pH 7)
    50 mM NaCl

Acknowledgments

This work was supported by funds from the United States Public Health Service (to B. S. K.), the Parker B. Francis Foundation (to T. T. B.), and the Infectious Disease Society of America (to G. M. G.). The University of Wisconsin-Madison Biophysics Instrumentation Facility, where the CD experiments were performed, was established by National Science Foundation Grant BIR9512577 and National Institutes of Health Grant RR13790.

References

  1. Brandhorst, T. T., Gauthier, G. M., Stein, R. A. and Klein, B. S. (2005). Calcium binding by the essential virulence factor BAD-1 of Blastomyces dermatitidis. J Biol Chem 280(51): 42156-42163.

简介

BAD-1是由两种类型的真菌Blastomyces dermatitidis(芽生菌病的病原体)产生的粘附素。 我们已经确定它对肝素具有亲和力,这可以解释其对毒力和人类免疫功能的影响,因为许多与免疫功能相关的细胞在其表面上具有肝素样部分。 该测定允许定量可溶性BAD-1和固定化肝素之间的结合。

材料和试剂

  1. 肝素 - 琼脂糖树脂(Sigma-Aldrich,目录号:H6508)(在使用前,将其在5体积的tricine缓冲液中洗涤3次以除去游离肝素)
  2. 10μgBAD-1 [根据Brandhorst等人(2005)的方法纯化]]
  3. 25微升可溶性医用级注射用肝素钠(50mg/ml)(Elkins-Sinn Inc)
  4. Tricine缓冲液(Sigma-Aldrich,目录号:T0377)(参见Recipes)

设备

  1. Accuspin micro17微量离心机(Thermo Fisher Scientific)
  2. Nanodrop ND1000分光光度计

程序

  1. 将100μl0.1mg/ml BAD-1的三羟甲基氨基甲烷缓冲液与琼脂糖肝素树脂(5μl床体积)在25℃温育30分钟,偶尔搅拌。
  2. 通过在Accuspin微量离心机中以7000xg离心1分钟使树脂珠粒化。
  3. 在Nanodrop ND1000分光光度计中分析含有BAD-1的上清液的样品在280nm的光密度。 将该读数与添加了tricine缓冲液代替肝素 - 琼脂糖珠的BAD-1的对照溶液进行比较。 假定吸光度的差异对于粘附在肝素树脂床上的BAD-1是线性的
  4. 通过在可溶性医用级肝素存在下在tricine缓冲液中重复步骤1-3进行结合抑制研究。 将肝素以各种浓度(0.01,0.1和1mg/ml - 在0.1mg/ml肝素和更高浓度下观察到显着抑制)加入到三羟甲基氨基甲烷结合缓冲液中。 校正基线吸光度的测量值以说明添加的肝素抑制剂的吸光度

食谱

  1. Tricine缓冲区
    20mM三羟甲基氨基甲烷(pH7) 50mM NaCl

致谢

这项工作得到了美国公共卫生局(B. S.K.),Parker B. Francis基金会(T.T.B.B。)和美国传染病学会(G.M.G.)的资金支持。 威斯康星大学 - 麦迪逊大学生物物理仪器设备,其中进行CD实验,由国家科学基金会授予BIR9512577和国立卫生研究院授予RR13790建立。

参考文献

  1. Brandhorst,T.T.,Gauthier,G.M.,Stein,R.A。和Klein,B.S。(2005)。 钙结合由 Blastomyces dermatitidis的基本毒力因子BAD-1。 J Biol Chem 280(51):42156-42163。
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Copyright: © 2014 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. Brandhorst, T. T. (2014). Native BAD-1 Binding to Heparin-agarose. Bio-protocol 4(7): e1095. DOI: 10.21769/BioProtoc.1095.
  2. Taguchi, Y., Mistica, A. M., Kitamoto, T. and Schätzl, H. M. (2013). Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. PLoS Pathog 9(6): e1003466.
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