Summary

人类S100A12及其用于免疫细胞刺激的Ion诱导寡聚物的纯化

Published: September 29, 2019
doi:

Summary

该协议描述了一种用于重组无标记标记的钙结合蛋白S100A12及其用于人类单核细胞刺激测定的由水因诱导的寡聚物的纯化方法。

Abstract

在此协议中,我们描述了一种从大肠杆菌培养中纯化人体钙结合蛋白S100A12及其由水因诱导的寡聚物以进行免疫细胞刺激的方法。该协议基于两步色谱策略,其中包括在一个海龙交换色谱柱上的蛋白质预纯化,以及随后对疏水性相互作用柱的抛光步骤。该策略以可管理的成本生产高纯度和产量的 S100A12 蛋白质。对于免疫细胞的功能检测,最终残留内毒素污染需要仔细监测和进一步清洁步骤,以获得无内毒素蛋白质。大多数内毒素污染可以通过电一孔交换色谱法排除。为了消耗残留污染,此协议描述了使用离心过滤器的去除步骤。根据可用的抗子强体 S100A12 可以排列成不同的同源体。为了研究结构和功能之间的关系,本方案进一步描述了S100A12蛋白的电处理,然后化学交联以稳定S100A12寡聚物及其随后通过尺寸排除分离相 色 谱 法。最后,我们描述了一种基于细胞的测定,它证实了纯化蛋白的生物活性,并证实了LPS的无制备。

Introduction

S100A12是一种钙结合蛋白,主要由人类粒细胞产生。蛋白质在(全身)炎症期间过度表达,其血清水平,特别是在(自动)炎症疾病,如全身性青少年特发性关节炎(sJIA),家族地中海热(FMF)或川崎病(KD)可以通知疾病活动和对治疗的反应。根据模式识别受体 (PRRs),如收费受体 (TPR),先天免疫系统可以通过病原体相关分子模式 (PAMPs)(如脂多糖 (LPS) 或损害相关分子模式 (DamPs;也称为”报警器”)。DAM是内源性分子,如细胞蛋白、脂质或核酸1。DAMP 功能很好地描述了钙素蛋白家族 S100A8/A9 和 S100A122的成员,据报告,它们还作为二价金属-氢合抗菌肽3、4 56.根据可用的抗气强度 S100A12 可以像 S100 系列的其他成员一样,排列成不同的同源体,直到最近,S100A12-寡头化对 PRR 相互作用(尤其是 TLR4)的影响还不得而知。

蛋白质的单体形式(92个氨基酸,10.2 kDa)由两个EF-手螺旋环螺旋结构组成,通过柔性连结器连接。C-终端 EF 手包含经典的 Ca2+绑定主题,而N端 EF 手则具有 S100 蛋白质特异性扩展环路结构(”伪 EF-手”),并揭示了减少的 Ca2+– 亲和力。S100A12 结合的 Ca2+可诱导蛋白质的 C-终点体发生重大构象变化,从而导致每个单体上接触疏水性斑块,并形成二分界面。因此,在生理条件下,由S100A12形成的最小四元结构是一种非共价二元结构(约21 kDa),其中单个单体处于反平行方向。当被安排为二聚体时,S100A12被报告给隔离Zn2+以及其他二价金属离子,例如,Cu2+具有高亲和力7。这些离子在S100A12二聚体界面由一个亚单位的氨基酸H15和D25以及抗平行的其他亚单元8、9、10的H89协调。虽然早期的研究表明,Zn2+负载 S100A12 可能诱导蛋白质的组织到同质四元体 (44 kDa) 和导致增加 Ca2+– 亲和力11,12,最近的金属滴定研究6建议Ca2+结合S100A12,以增加蛋白质对Zn2+的亲和力。一旦 S100A12 EF 指针被 Ca2+完全占用,则额外的 Ca2+被认为在二聚物之间结合,触发六项机形成(约 63 kDa)。六甲四聚体结构的结构与四合体结构明显不同。建议四联体接口被中断,产生新的二聚体-二聚体接口,有利于六项机形成10。S100A12几乎完全由人类粒细胞表达,约占所有细胞蛋白的5%。在其DAMP功能S100A12在历史上被描述为高级糖化最终产品(RAGE)的多配体受体的激动剂,然后称为细胞外新发现的RAGE结合蛋白(EN-RAGE)14。尽管我们早先报告生化S100A12对RAGE和TLR415结合,但我们最近证明人类单核细胞以TLR4依赖方式对S100A12刺激作出反应。这需要将S100A12排列到其Ca2+/Zn2+-诱导的六甲体结构16中。

在这里,我们描述了重组人类S100A12及其免疫细胞刺激的孔诱导寡聚物的纯化程序16,17。这是基于两步色谱策略,最初包括一个电一体交换柱,以分离和浓缩蛋白质,并去除散装污染(例如,内毒素/脂多糖)18。电一交换色谱树脂根据不同的净表面电荷分离蛋白质。对于酸性蛋白质,如S100A12(等电点5.81),pH值为8.5的缓冲系统和强的黄极交换树脂可产生良好的分离。结合蛋白用高盐缓冲梯度洗脱。随着洗脱缓冲液中离子强度负离子的增加,与树脂表面的蛋白质竞争。蛋白质根据其净电荷单独洗出,因此,本文描述的缓冲液允许分离和浓缩过度表达的S100A12蛋白。由于脂多糖中的带负电荷,这些分子还与阴极交换树脂结合。然而,其较高的净电荷导致在应用高盐梯度的后期洗脱。为抛光目的介绍了纯化程序的第二步。这利用S100A12的钙结合能力,并去除疏水相互作用柱上剩余的杂质。S100A12 的钙结合会导致形成变化和在蛋白质表面接触疏水性斑块。在这种情况下,S100A12 与树脂的疏水表面相互作用。当EDTA的钙合合后,这种相互作用被逆转。在离子(尤其是钙和锌)存在的情况下,S100A12 可排列成同质寡聚物。为了研究不同寡聚物的结构-功能关系,我们用化学交联器稳定了二聚体、四甲和六甲重孔S100A12,并在尺寸排除色谱柱上分离了复合物。最后,对纯化蛋白及其电子诱导寡聚物的功能和生物活性进行了分析,对S100A12和LPS刺激的单核细胞的细胞因子释放进行了比较。

到目前为止,已经描述了各种净化S100A12的方法。例如,Jackson等人19日通过一个电子交换柱和随后的尺寸排除色谱,发表了一种纯化协议。在大小排除列上纯化抛光可产生良好的效果,但由于加载量有限等原因,可伸缩性不够灵活。由Kiss等人20号发表的另一种方法,将通过Ni2+亲和柱纯化标记蛋白描述为第一步,然后是酶裂解,以去除标记和进一步纯化步骤。与上述研究19、20相反,本协议中描述的所产生的蛋白质被确定为免疫细胞实验。因此,细菌培养的残留内毒素污染是一个挑战。虽然到目前为止已经描述了不同的内毒素去除方法,但没有统一的方法对任何给定的蛋白质溶液21,22同样有效。

总之,我们的协议将细菌系统中无标签表达的优点与高效的内毒素去除和纯蛋白的高产量相结合。

Protocol

注:有关准备缓冲液和库存解决方案,请参阅补充表 1。 1.大肠杆菌中的蛋白质表达 克隆 将无标记的人类S100A12(NCBI参考序列:NP_005612.1)克隆成细菌表达载体pET11b。为了表达蛋白质,将结构转换为大肠杆菌BL21(DE3)。 文化 通过在 14 mL 圆底管中接种 1mL 生长培养基(LB …

Representative Results

在AIEX列(图1A-C)和随后的钙依赖性HIC(图2A,B)上预纯后,获得高纯蛋白(图2C)。此外,内毒素的测量显示LPS去除成功。AIEX 之后的 LPS 含量在高于测定检测限值(即高于 500 EU/mL)的 1:10 稀释度下测量。通过 50 kDa 过滤单元进行第一次过滤后,LPS 含量降至 1 欧/mL。在使用 3 kDa 滤光片和通过 50 kDa 进行?…

Discussion

在此协议中,我们描述了人类S100A12的无标记细菌表达及其纯化以及分离成不同的由水因诱导的寡聚物,用于免疫细胞刺激。与发表论文的S100A12蛋白质纯化8,23,24,使用高CaCl2(25 mM)在疏水-相互作用色谱是对我们的知识的独特。应用浓度为 1 到 5 mM 的几种方案确实会产生纯蛋白质,但我们观察到,使用 25 mM CaCl2</…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项研究得到了蒙斯特大学医学院(KE121201至C.K.)和德国研究基金会(DFG,Fo354/3-1到D.F.)的校内创新医学研究项目的资助。

Materials

pET11b vector Novagen    
BL21(DE3) competent E. coli New England Biolabs C2527  
       
100 x Non-essential amino acids Merck K 0293
25% HCl Carl Roth X897.1
4−20% Mini-PROTEAN TGX Protein Gels BioRad 4561093
Ampicillin sodium salt Carl Roth HP62.1
BS3 (bis(sulfosuccinimidyl)suberate) – 50 mg ThermoFisher Scientific 21580
Calciumchlorid Dihydrat Carl Roth 5239.1
Coomassie Briliant Blue R250 Destaining Solution BioRad 1610438
Coomassie Briliant Blue R250 Staining Solution BioRad 1610436
EasySep Human Monocyte Enrichment Kit Stemcell 19059 Magnetic negative cell isolation kit
EDTA disodium salt dihydrate Carl Roth 8043.1
EGTA Carl Roth 3054.3
EndoLISA Hyglos 609033 Endotoxin detection assay
Endotoxin-Free Ultra Pure Water Sigma-Aldrich TMS-011-A Ultrapure water for preparation of endotoxin-free buffers
EndoTrap red Hyglos 321063 Endotoxin removal resin
FBS (heat-inactivated) Gibco 10270
HBSS, no calcium, no magnesium ThermoFisher Scientific 14175053
Hepes Carl Roth 9105
Hepes (high quality, endotoxin testet) Sigma-Aldrich H4034
hTNF-alpha – OptEia ELISA Set BD 555212
IPTG (isopropyl-ß-D-thiogalactopyranosid) Carl Roth CN08.1
L-Glutamine (200 mM) Merck K 0282
LB-Medium Carl Roth X968.1
Lipopolysaccharides from E. coli O55:B5 Merck L6529
Pancoll, human PAN Biotech P04-60500 Separation solution (density gradient centrifugation)
Penicillin/Streptomycin (10.000 U/ml) Merck A 2212
Phenyl Sepharose High Performance GE Healthcare 17-1082-01 Resin for hydrophobic interaction chromatography
Polymyxin B Invivogen tlrl-pmb
Protease inhibitor tablets Roche 11873580001
Q Sepharose Fast Flow GE Healthcare 17-0510-01 Resin for anion-exchange chromatography
RoboSep buffer Stemcell 20104 Cell separation buffer (section 5.1.4)
RPMI 1640 Medium Merck F 1215
Sodium chloride (NaCl) Carl Roth 3957.2
Sodium hydroxide Carl Roth P031.1
Tris Base Carl Roth 4855.3
Zinc chloride Carl Roth T887
Labware
0,45 µm syringe filter Merck SLHA033SS
14 mL roundbottom tubes BD 352059
2 L Erlenmyer flask Carl Roth LY98.1
24 well suspension plates Greiner 662102
5 L measuring beaker Carl Roth CKN3.1
50 mL conical centrifuge tubes Corning 430829
50 mL high-speed centrifuge tubes Eppendorf 3,01,22,178
Amicon Ultra-15 Centrifugal Filter Unit MWCO 3 kDa Merck UFC900324
Amicon Ultra-15 Centrifugal Filter Unit MWCO 50 kDa Merck UFC905024
Culture dish (100 mm) Sarstedt 83.3902
Dialysis Tubing Closures Spectrum 132738
EasySep magnet 'The Big Easy` Stemcell 18001
Fraction collector tubes 5 mL Greiner 115101
Lumox film, 25 µm, 305 mm x 40 m Sarstedt 94,60,77,316 Film for monocyte culture plates
Spectra/Por Dialysis Membrane (3.5 kDa) Spectrum 132724
Steritop filter unit Merck SCGPT01RE
Equipment
37 °C Incubator (with shaking) New Brunswick Scientific Innova 42
ÄKTA purifier UPC 10 GE Healthcare FPLC System
Fraction collector GE Healthcare Frac-920
Centrifuge (with rotor A-4-81) Eppendorf 5810R
Fixed angle rotor Eppendorf F-34-6-38
Mini Protean Tetra Cell BioRad 1658000EDU
NanoPhotometer Implen P330
Sonicator Brandelin UW2070
Fluorescence reader Tecan infinite M200PRO
pH meter Knick 765

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Citazione di questo articolo
Fuehner, S., Foell, D., Kessel, C. Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation. J. Vis. Exp. (151), e60065, doi:10.3791/60065 (2019).

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