用于神经元细胞贩运研究的管子中纯化和直接单生物素重组BDNF的改进协议
1Department of Physiology, Faculty of Biological Sciences,Pontificia Universidad Católica de Chile, 2Department of Molecular and Cellular Biology, Faculty of Biological Sciences, Center for Aging and Regeneration (CARE UC),Pontificia Universidad Católica de Chile, 3Institute of Biomedical Sciences. Faculty of Medicine and Faculty of Life Sciences,Universidad Andrés Bello, 4Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and UK Dementia Research Institute at UCL,University College London Campus
Summary
含有 Avi 序列 （BDNFAvi） 的重组 BDNF 以经济高效的方式在 HEK293 细胞中生产，并通过亲和色谱进行纯化。然后，BDNFavi 与管中的酶 BirA 直接单生物素化。与市售的BDNF相比，BDNFavi和单生物素化BDNFavi保留了其生物活性。
Abstract
含有 Avi 序列 （BDNFAvi） 的重组 BDNF 在 HEK293 细胞中生产，然后通过亲和力色谱进行经济高效地纯化。一种可重复的协议被开发成直接单生物素BDNFAvi与酶BirA在管中。在这个反应中，单生物素化BDNFAvi保持其生物活性。
神经营养素是靶源生长因子，在神经元发育和维持中起着一定的作用。它们需要沿着内分泌通路的快速传输机制，以便在不同的神经元隔间之间进行远距离信号。开发分子工具，研究神经营养素的贩运，使这些蛋白质在细胞使用体内记录精确跟踪。在该协议中，我们为单生物基化BDNF的生产开发了一个优化且经济高效的程序。含有生物素可 avi 序列 （BDNFAvi） 的重组 BDNF 变种在微克范围内的 HEK293 细胞中产生，然后使用亲和力色谱在易于分量的程序中纯化。然后，纯化的BDNF可以通过与管中的BirA酶直接体外反应来均匀地进行单生物素化。单生物基化BDNF（mbtBDNF）的生物活性可以与与不同荧光团结合的链球菌结合。BDNFAvi和mbtBDNF分别通过利用西方印迹检测下游磷酸化靶点和活化转录因子CREB来保持其生物活性。使用链球菌素量子点，我们能够可视化 mbtBDNF 内化伴随激活 CREB，该激活与磷-CREB特异性抗体一起检测。此外，与链球菌素量子点结合的mbtBDNF适用于微流体室中生长的皮质神经元的逆行性传输分析。因此，在管生产mbtBDNF是一个可靠的工具，研究生理信号内位动力学和神经元的贩运。
Introduction
神经元是神经系统的功能单位，具有复杂和专门的形态，允许突触通信，因此，产生协调和复杂的行为，以回应不同的刺激。神经元投影，如树突和轴突是参与神经元交流的关键结构特征，神经营养素是决定其形态和功能1的关键参与者。神经营养素是一个分泌生长因子的家族，包括NGF、NT-3、NT-4和脑源神经营养因子（BDNF）2。在中枢神经系统（CNS），BDNF参与不同的生物过程，包括神经传学，树突状，树突脊柱的成熟，长期强权，等等3，4。3,因此，BDNF在调节神经元功能方面起着至关重要的作用。
不同的细胞过程调节BDNF动力学和功能。在神经元表面，BDNF结合肌蛋白酶受体激酶B（TrkB）和/或p75神经营养素受体（p75）。BDNF-TrkB和BDNF-p75复合物被内分泌和排序在不同的内分泌细胞器5，6，7，8。,6,7,8BDNF/TrkB复合物的细胞内贩运需要在不同的神经元,电路9、10、11,中发出正确的BDNF信号。10因此，深入了解BDNF贩运动态及其在病理生理过程中的变化对于了解BDNF在健康和疾病中的信号至关重要。开发新的和特定的分子工具来监测这一过程将有助于推动这一领域向前发展，并更好地掌握所涉及的监管机制。
有几个工具可用于研究BDNF贩运神经元。常用的方法涉及转染重组BDNF与荧光分子标记，如绿色荧光蛋白（GFP）或单体荧光红移变种的GFP mCherry12，13。,13然而，BDNF过度表达的一个主要缺点是，它消除了提供这种神经营养素的已知浓度的可能性。此外，它可能会导致细胞毒性，掩盖了结果14的解释。另一种策略是表位标记的TrkB的转染，如旗-TrkB。这种方法允许研究TrkB内化动力学15，但它也涉及转染，这可能会导致TrkB功能和细胞毒性的改变。为了克服这些方法障碍，16，17，开发了含有Avi序列（BDNFAvi）的NGF和BDNF的重组变种，这种变异可以由生物素-利加酶BirA单生物素化。生物基化重组BDNF可与不同的链球菌结合工具耦合，这些工具包括荧光团、珠子、顺磁纳米粒子等，用于检测。在活细胞成像方面，量子点（QD）已成为常用的荧光团，因为它们具有单粒子跟踪的可取特性，如与小分子荧光团18相比，亮度增加，光漂白的抵抗力增强。
使用BDNFAvi的单生物基化BDNF（mbtBDNF）的产生是通过对驱动BDNFAvi和BirA表达的质粒进行共生共进，然后通过亲和力色谱纯化重组蛋白，每20mL的 HEK293条件培养基17产生1-2μg的BDNF。在这里，我们建议修改该协议，允许BDNFAvi纯化从500 mL的 HEK293 条件介质，寻求最大限度地提高蛋白质回收在色谱柱为基础的协议，以方便操作。用过的转染剂聚乙烯胺 （PEI） 可确保在不牺牲转染产量的情况下采用经济高效的方法。单生物素化步骤已适应体外反应，以避免与共创相关的并发症，并确保BDNF的均匀标记。mbtBDNF的生物活性通过西方的印迹和荧光显微镜实验得到证明，包括pCREB的活化和活细胞成像，以研究BDNF在微流体室中的逆行性。使用该协议允许优化，高产生产同质单生物基化和生物活性BDNF。
Protocol
所有实验都是按照智利国家科学和技术研究委员会（智利国家科学技术研究委员会）核准的准则进行的。本研究中使用的议定书得到智利大学生物安全和生物伦理及动物福利委员会的批准。涉及脊椎动物的实验得到智利大学生物伦理和动物福利委员会的批准。 注：以下协议旨在从 HEK293 细胞中产生的 500 mL 条件介质的总体积中纯化 BDNFAvi。为净化BDNFAvi而生产并加工的有条件介?…
Representative Results
使用基于色谱柱的协议可以处理大量 HEK293 条件介质。在图1中，显示了从500mL的调节介质中纯化BDNFAvi的结果。从Ni-NTA agarose珠连续洗脱BDNFAvi产生BDNFAvi浓度下降（图1A）。经过四次连续洗脱（每次持续15分钟），珠子捕获的大多数BDNF被恢复。Eluates 的浓度范围为 6 至 28 ng/μL，总产量约为 BDNFAvi 的 60 μg（表 1）。然后，由BirA-GST调解的体外反应…
Discussion
本文根据宋和合作者17的工作，描述了在基于亲和力色谱的工艺中生产和纯化mbtBDNF的优化方法。优化包括使用具有成本效益的转染试剂 （PEI），同时保持更昂贵的转染方法（如脂氨基胺）的效率。这种优化可显著降低协议成本，实现可扩展性，同时保持高成本效益。该协议还包括易用性考虑，包括冻结有条件介质长达2个月。这些优化使程序适应每个实验室的需求，提高成本效?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢方德西特 （1171137） （FCB）， 巴塞尔科技卓越中心 （AFB 170005） （FCB）， 千禧年核心 （P07/011-F（FCB）、威康信托高级调查员奖（107116/Z/15/Z）和英国痴呆症研究所基金会奖（GS）。这项工作得到了UC微镜协会（UMA UC）的支持。
Materials
| 2 way stopcock | BioRad | 7328102 | Chromatography apparatus component |
| 2-mercaptoethanol | Sigma | M6250 | BDNF elution buffer |
| Acrylamide/Bisacrylamide | BioRad | 1610154 | SDS-PAGE gel preparation |
| Amicon Ultra-15 10K | Millipore | UFC901024 | BDNF concentration |
| Ammonium Persulfate | Sigma | A9164 | SDS-PAGE gel preparation |
| anti B-III-Tubulin antibody | Sigma | T8578 | Western blot assays for BDNF biological activity detection |
| anti BDNF antibody | Alomone | AGP-021 | Western blot assays for BDNF quantification |
| anti BDNF antibody | Alomone | ANT-010 | Western blot assays for BDNF quantification |
| Anti ERK antibody | Cell Signaling | 9102 | Western blot assays for BDNF biological activity detection |
| anti pCREB antibody (S133) | Cell Signaling | 9198 | Western blot assays for BDNF biological activity detection |
| anti pERK antibody (T202, Y204) | Cell Signaling | 4370 | Western blot assays for BDNF biological activity detection |
| anti pTrkB antibody (Y515) | Abcam | ab109684 | Western blot assays for BDNF biological activity detection |
| Antibiotic/Antimycotic | Gibco | 15240-062 | HEK293 maintenance |
| ATP | Sigma | A26209 | BDNF monobiotinylation buffer |
| B-27 Supplement | Gibco | 17504-044 | Neuron maintenance |
| Bicine | Sigma | B3876 | BDNF monobiotinylation buffer |
| BirA-GST | BPS Bioscience | 70031 | Enzyme for BDNF AviTag monobiotinylation |
| Bovine Fetal Serum | HyClone | HC.SH30396.02 | HEK293 maintenance |
| Bovine Serum Albumin | Jackson ImmunoResearch | 001-000-162 | BDNF buffer modification component, blocking buffer for western blot and immunofluorescence |
| D-Biotin | Sigma | B4639 | BDNF monobiotinylation buffer |
| Dithiothreitol | Invitrogen | 15508-013 | |
| DMEM High Glucose Medium | Gibco | 11965-092 | Neuron seeding |
| DMEM Medium | Gibco | 11995-081 | HEK293 maintenance |
| Econo Column Funnel | BioRad | 7310003 | Chromatography apparatus component |
| EDTA | Merck | 108418 | |
| EZ-ECL Kit | Biological Industries | 1633664 | Protein detection by western blotting |
| Glutamax | Gibco | 35050-061 | Neuron and HEK293 maintenance |
| Glycerol | Merck | 104094 | BDNF elution buffer, lysis buffer for western blot assays |
| Hettich Rotina 46R Centrifuge | Hettich | Discontinued | Centrifuge used for clearing the medium of debris |
| Hettich Universal 32R Centrifuge | Hettich | Discontinued | Centrifuge used for protein concentrator centrifugation |
| Horse Serum | Gibco | 16050-122 | Neuron seeding |
| ImageQuant LAS 500 | GE Healthcare Life Sciences | 29005063 | Western blot image acquisition |
| Imidazole | Sigma | I55513 | BDNF buffer modification component |
| KCl | Winkler | BM-1370 | PBS component |
| KH2PO4 | Merck | 104873 | PBS component |
| Laminin | Invitrogen | 23017-015 | Cover coating for compartmentalized neurons |
| Luer Tubing Adaptor | BioRad | 7323245 | Chromatography apparatus component |
| Luminata™ Forte Western HRP Substrate | Millipore | WBLUF0100 | Protein detection by western blotting |
| Mg(CH3COO)2 | Merck | 105819 | BDNF monobiotinylation buffer |
| Mowiol 4-88 | Calbiochem | 475904 | Mounting reagent for immunofluorescence assays |
| MyOne C1 Streptavidin Magnetic Beads | Invitrogen | 65001 | Biotinylation verification |
| Na2HPO4 | Merck | 106586 | BDNF buffer modification component |
| NaCl | Winkler | BM-1630 | PBS component, BDNF buffer modification component |
| NaH2PO4 | Merck | 106346 | BDNF buffer modification component |
| Neurobasal Medium | Gibco | 21103-049 | Neuron maintenance |
| Ni-NTA Agarose Beads | Qiagen | 30210 | BDNF AviTag purification |
| Nikon Ti2-E | Nikon | Microscope for fluorescence imaging | |
| Nitrocellulose Membrane | BioRad | 1620115 | Protein transfer for western blotting |
| ORCA-Flash4.0 V3 Digital CMOS camera | Hamamatsu | C13440-20CU | Camera for epifluorescence imaging |
| P8340 Protease Inhibitor Cocktail | Sigma | P8340 | BDNF buffer modification component |
| Paraformaldehyde | Merck | 104005 | Fixative for immunofluorescence assays |
| Penicillin/Streptomycin | Gibco | 15140-122 | Neuron maintenance |
| Poli-D-Lysine | Corning | DLW354210 | Cover coating for compartmentalized neurons |
| Poli-L-Lysine | Millipore | P2363 | Cover coating for non-compartmentalized neurons |
| Poly-Prep Chromatography Column | BioRad | 7311550 | Chromatography apparatus component |
| Polyethyleneimine 25K | Polysciences Inc. | PLY-0296 | HEK293 transfection |
| Quantum Dots 655 streptavidin conjugate | Invitrogen | Q10121MP | Monobiotinylated BDNF AviTag label for live and fixed cell experiments |
| Saponin | Sigma | S4521 | Detergent for immunofluorescence assays |
| Sucrose | Merck | 107687 | |
| Syldgard 184 silicone elastomer base | Poirot | 4019862 | Microfluidic chamber preparation |
| TEMED | Sigma | T9281 | SDS-PAGE gel preparation |
| Tris | Winkler | BM-2000 | Lysis buffer component |
| Triton X100 | Merck | 108603 | Cell permeabilization in immunofluorescence and western blot assays |
| Trypsin-EDTA 0.5% | Gibco | 15400-054 | HEK293 passaging |
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Citer Cet Article
Stuardo, N., Moya-Alvarado, G., Ramírez, C., Schiavo, G., Bronfman, F. C. An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons. J. Vis. Exp. (161), e61262, doi:10.3791/61262 (2020).