从海蛾中亲和纯化纤维蛋白溶解酶
Summary
在这里，我们提出了一种来自 海蜥蜴 纤维蛋白溶解酶的亲和纯化方法，该方法简单、廉价且高效。
Abstract
海蕊纤溶酶（sFE）是一种新型纤溶剂，既能将纤溶酶原激活成 纤 溶酶，又能直接降解纤维蛋白，与传统溶栓剂相比具有很大的优势。然而，由于缺乏结构信息，所有sFE的纯化程序都基于多步色谱纯化，过于复杂和昂贵。本文首次基于sFE的晶体结构开发了sFE亲和纯化方案;它包括粗样品和赖氨酸/精氨酸-琼脂糖基质亲和色谱柱的制备、亲和纯化和纯化 sFE 的表征。按照该方案，一批sFE可以在1天内纯化。此外，纯化的sFE的纯度和活性分别增加到92%和19，200 U/mL。因此，这是一种简单、廉价且高效的sFE纯化方法。该方案的开发对于sFE和其他类似药物的进一步利用具有重要意义。
Introduction
血栓形成是对公共卫生的主要威胁，尤其是在 Covid-19 全球大流行之后1，2。临床上，许多纤溶酶原激活剂（PAs），如组织型纤溶酶原激活剂（tPA）和尿激酶（UK），已被广泛用作溶栓药物。PA可以将患者的纤溶酶原激活为活性纤溶酶以降解纤维蛋白。因此，它们的溶栓效率受到患者纤溶酶原状态3，4的严重限制。纤维蛋白溶解剂，如金属蛋白酶纤溶酶和丝氨酸纤溶酶，是另一种临床溶栓药物，还包括纤溶酶（FE），如纤溶酶，可直接溶解凝块，但被各种纤溶酶抑制剂迅速灭活5。随后，报道了一种新型的纤维蛋白溶解剂，它不仅可以通过将纤溶酶原激活为纤溶酶，还可以直接降解纤维蛋白6-来自古代花生蠕虫Sipunculus nudus（sFE）的纤维蛋白溶解酶6来溶解血栓6。与传统的溶栓药物相比，这种双功能赋予了sFE其他优势，特别是在纤溶酶原状态异常方面。与其他双功能纤维蛋白溶解剂7，8，9相比，sFE在药物开发方面表现出几个优势，包括安全性，特别是对于口服药物。这是因为海蜥的生物安全性和生物相容性已经确立10.
与从微生物、蚯蚓和蘑菇中分离的其他天然纤维蛋白溶解剂类似，从裸蚯蚓中纯化sFE非常复杂，包括组织匀浆、硫酸铵沉淀、脱盐、阴离子交换色谱、疏水相互作用色谱和分子筛等多个阶段10，11，12.这样的净化系统不仅要依靠熟练的技能和昂贵的材料，还需要几天的时间才能完成整个过程。因此，一个简单的sFE纯化方案对sFE的进一步发展具有重要意义。幸运的是，两个sFE晶体（PDB：8HZP;PDB：8HZO）已成功获得（见补充文件1和补充文件2）。通过结构分析和分子对接实验，我们发现sFE的催化核心可以特异性地与含有精氨酸或赖氨酸残基的靶标结合。
本文首次提出了一种基于sFE晶体结构的亲和纯化系统。通过遵循该方案，可以在单个亲和纯化阶段从粗提取物中纯化高纯度和高活性sFE。这里开发的方案不仅对sFE的大规模制备很重要，而且可用于纯化其他纤维蛋白溶解剂。
Protocol
1. 准备 样品处理仔细解剖新鲜的 裸粒链球菌 （100克）并收集肠道及其内部液体。 加入 300 mL Tris-HCl 缓冲液（0.02 M，pH 7.4）进行均质化（1，000 rpm，60 s）。 将匀浆冻融3倍。 离心样品（10，956× g，0.5小时，4°C）并收集上清液。将样品储存在4°C直至进一步使用。 蛋白质沉淀将上清液与饱和硫酸铵溶液（九体积?…
Representative Results
按照该方案，提取粗组织裂解物，建立精氨酸-琼脂糖基质和赖氨酸-琼脂糖基质亲和色谱柱，得到纯化的sFE，分别用SDS-PAGE和纤维蛋白板测定纯化sFE的纯度和纤维蛋白溶解活性。 离心后，收集的上清液为透明的棕褐色粘稠液体。当该上清液与饱和硫酸铵溶液（九体积）混合时开始沉淀。静置12 h后，在管底形成重沉淀。当用Tris-HCl缓冲液重悬时，蛋白质沉淀迅速消失并溶解在缓?…
Discussion
由于sFE的确切基因序列不可用，目前使用的sFE是从新鲜 的S. nudus14中提取的。此外，文献报道的sFE纯化程序复杂且昂贵，因为它们基于sFE的一些一般特征，如分子量，等电点，离子强度和极性15，16。迄今为止，尚未报道sFE的亲和纯化方案。本研究基于sFE晶体结构知识，成功开发了sFE亲和纯化方案。与报道的纯化方法相比，该亲?…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究由厦门市科技局（3502Z20227197）和福建省科技局（编号：2019J01070，No.2021Y0027）资助。
Materials
| 30% Acrylamide-Bisacrylamide (29:1) | Biosharp | ||
| 2-Mercaptoethanol | Solarbio | ||
| Agarose G-10 | Biowest | ||
| Ammonium persulfate | SINOPHARM | ||
| Ammonium sulfate | SINOPHARM | ||
| Arginine-Sepharose 4B | Solarbio | Arginine-agarose matrix | |
| Bromoxylenol Blue (BPB) | Solarbio | ||
| Fast Silver Stain Kit | Beyotime | ||
| Fibrinogen | Merck | ||
| Glycine | Solarbio | ||
| Hydrochloric acid | SINOPHARM | ||
| Kinase | RHAWN | ||
| Lysine-Sepharose 4B | Solarbio | Lysine-agarose matrix | |
| N,N,N',N'-Tetramethylethylenediamine (TEMED) | Sigma-Aldrich | ||
| Prestained Color Protein Marker (10-170 kD) | Beyotime | ||
| Sodium chloride | SINOPHARM | ||
| Sodium Dodecyl Sulfonate (SDS) | Sigma-Aldrich | ||
| Sodium hydroxide | SINOPHARM | ||
| Thrombin | Meilunbio | ||
| Tris(Hydroxymethyl) Aminomethane | Solarbio | ||
| Tris(Hydroxymethyl) Aminomethane Hydrochloride | Solarbio | ||
| Equipment | |||
| AKT Aprotein Purification System pure | GE | ||
| Automatic Vertical Pressure Steam Sterilizer MLS-3750 | SANYO | ||
| Chemiluminescence Imaging System | GE | ||
| Constant Flow Pump BT-100 | QITE | ||
| Constant Temperature Incubator | JINGHONG | ||
| Desktop Refrigerated Centrifuge 3-30KS | SIGMA | ||
| DHG Series Heating and Drying Oven DGG-9140AD | SENXIN | ||
| Electric Glass Homogenizer DY89-II | SCIENTZ | ||
| Electronic Analytical Balance | DENVER | ||
| Electro-Thermostatic Water Bath DK-S12 | SENXIN | ||
| Horizontal Decolorization Shaker | Kylin-Bell | ||
| Ice Machine AF 103 | Scotsman | ||
| KQ-500E Ultrasonic Cleaner | ShuMei | ||
| Magnetic Stirrer | Zhi wei | ||
| Micro Refrigerated Centrifuge H1650-W | Cence | ||
| Microwave Oven | Galanz | ||
| Milli-Q Reference | Millipore | ||
| Pipettor | Thermo Fisher Scientific | ||
| Precision Desktop pH Meter | Sartorious | ||
| Small-sized Vortex Oscillator | Kylin-Bell | ||
| Vertical Electrophoresis System | Bio-Rad | ||
| Consumable Material | |||
| 200 µL PCR Tube (200 µL) | Axygene | ||
| Centrifuge Tube (1.5 mL) | Biosharp | ||
| Centrifuge Tube (5 mL) | Biosharp | ||
| Centrifuge Tube (50 mL) | NEST | ||
| Centrifuge Tube (7 mL) | Biosharp | ||
| Culture Dish (60 mm) | NEST | ||
| Filter Membrane (0.22 µm) | Millex GP | ||
| Parafilm | Bemis | ||
| Pipette Tip (1 mL ) | KIRGEN | ||
| Pipette Tip (10 µL) | Axygene | ||
| Pipette Tip (200 µL) | Axygene | ||
| Special Indicator Paper | TZAKZY | ||
| Ultra Centrifugal Filter Unit (15 mL 3 KDa) | Millipore | ||
| Ultra Centrifugal Filter Unit (4 mL 3 KDa) | Millipore | ||
| Universal pH Indicator | SSS Reagent |
Referências
- Rosell, A., et al. Patients with COVID-19 have elevated levels of circulating extracellular vesicle tissue factor activity that is associated with severity and mortality-brief report. Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (2), 878-882 (2021).
- Schultz, N. H., et al. Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination. The New England. Journal of Medicine. 384 (22), 2124-2130 (2021).
- von Kaulla, K. N. Urokinase-induced fibrinolysis of human standard clots. Nature. 184 (4695), 1320-1321 (1959).
- Van de Werf, F., et al. Coronary thrombolysis with tissue-type plasminogen activator in patients with evolving myocardial infarction. The New England Journal of Medicine. 310 (10), 609-613 (1984).
- Schaller, J., Gerber, S. S. The plasmin-antiplasmin system: structural and functional aspects. Cellular and Molecular Life Sciences. 68 (5), 785-801 (2011).
- Ge, Y. -. H., et al. A novel antithrombotic protease from marine worm Sipunculus nudus. International Journal of Molecular Sciences. 19 (10), 3023 (2018).
- Liu, X., et al. Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus. Journal of Microbiology and Biotechnology. 24 (2), 245-253 (2014).
- Choi, J. -. H., Sapkota, K., Kim, S., Kim, S. -. J. Starase: A bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential. Biochimie. 105, 45-57 (2014).
- Liu, H., et al. A novel fibrinolytic protein From Pheretima vulgaris: purification, identification, antithrombotic evaluation, and mechanisms investigation. Frontiers in Molecular Biosciences. 8, 772419 (2022).
- Wu, Y., et al. Antioxidant, hypolipidemic and hepatic protective activities of polysaccharides from Phascolosoma esculenta. Marine Drugs. 18 (3), 158 (2020).
- . Preparation and application of natural fibrinolytic enzyme from peanut worm Available from: https://patents.google.com/patent/CN109295042A/en (2019)
- Li, W., Yuan, M., Wu, Y., Xu, R. Identification of genes expressed differentially in female and male gametes of Sipunculus nudus. Aquaculture Research. 51 (9), 3780-3789 (2020).
- Ossipow, V., Laemmlii, U. K., Schibler, U. A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis. Nucleic Acids Research. 21 (25), 6040-6041 (1993).
- Hsu, T., Ning, Y., Gwo, J., Zeng, Z. DNA barcoding reveals cryptic diversity in the peanut worm Sipunculus nudus. Molecular Ecology Resources. 13 (4), 596-606 (2013).
- Abiko, Y., Iwamoto, M., Shimizu, M. Plasminogen-plasmin system. I. Purification and properties of human plasminogen. The Journal of Biochemistry. 64 (6), 743-750 (1968).
- Abiko, Y., Iwamoto, M., Shimizu, M. Plasminogen-plasmin system. II. Purification and properties of human plasmin. The Journal of Biochemistry. 64 (6), 751-757 (1968).
- Wiman, B. Affinity-chromatographic purification of human α2-antiplasmin. The Biochemical Journal. 191 (1), 229-232 (1980).
- Sandbjerg Hansen, M., Clemmensen, I. Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors. The Biochemical Journal. 187 (1), 173-180 (1980).
- Pietrocola, G., Rindi, S., Nobile, G., Speziale, P. Purification of human plasma/cellular fibronectin and fibronectin fragments. Fibrosis. 1627, 309-324 (2017).
- Nabiabad, H. S., Yaghoobi, M. M., Javaran, M. J., Hosseinkhani, S. Expression analysis and purification of human recombinant tissue type plasminogen activator (rt-PA) from transgenic tobacco plants. Preparative Biochemistry and Biotechnology. 41 (2), 175-186 (2011).
- Shearin, T. V., Pizzo, S. V., Gonzalez-Gronow, M. Molecular abnormalities of human plasminogen isolated from synovial fluid of rheumatoid arthritis patients. Journal of Molecular Medicine. 75 (5), 378-385 (1997).
Citar este artigo
Tang, M., Lin, H., Hu, C., Yan, H. Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus. J. Vis. Exp. (196), e65631, doi:10.3791/65631 (2023).