如何表达具有活性的全长膜蛋白？
解决方案摘要
跨膜蛋白作为细胞膜的重要组成部分, 在物质运输、信号转导和细胞间识别等多种细胞功能中发挥着重要作用。其功能异常往往会导致疾病的发生，这使它们成为理想的药物作用靶点。目前以跨膜蛋白为药物靶点占现阶段己知药物靶点的 60% 以上。而针对抗体药靶点，膜蛋白几乎占 90% 以上。为助力药物研发，解决跨膜蛋白靶点开发过程中抗原制备的难题，缔码生物专门搭建了多个跨膜蛋白全长表达平台。
产品配置单
解决方案详情
跨膜蛋白及药物研发
跨膜蛋白作为细胞膜的重要组成部分, 在物质运输、信号转导和细胞间识别等多种细胞功能中发挥着重要作用。其功能异常往往会导致疾病的发生，这使它们成为理想的药物作用靶点。目前以跨膜蛋白为药物靶点占现阶段己知药物靶点的 60% 以上。而针对抗体药靶点，膜蛋白几乎占 90% 以上。
跨膜蛋白在物质运输、信号转导和细胞间识别等多种细胞功能中发挥重要作用
抗原制备
尽管有着重大的意义，针对跨膜蛋白的药物开发仍然具有很大的挑战性，主要是因为跨膜蛋白的疏水结构，使得其在体外很难以可溶性蛋白形式保持其天然构象，同时全长多跨膜蛋白通常表达水平较低，这些都是跨膜蛋白表达的难点所在。如何获得最为天然构象和具有功能活性的膜蛋白是开发这一类靶点抗体药物的核心所在。目前针对跨膜蛋白靶点，其抗原开发策略及相关问题如下：
表达胞外结构域（ECD）：表达胞外 N 端或 LOOP 区结构域的方法简单，并且相对而言有较高的表达量，是行之有效的方法之一。但是对于多跨膜蛋白而言，表达蛋白胞外区的方式很难模拟蛋白的天然构象，可能造成一些抗原表位（比如构象表位）的遗失，也有可能对蛋白活性有一定的影响。
全细胞：全细胞免疫被广泛用于针对跨膜蛋白的抗体制备。目标膜蛋白保持了自己的天然构象和修饰，具有高免疫原性，不需要复杂的纯化步骤。然而，全细胞成分较复杂，包含胞浆等多种杂蛋白组分，容易产生针对该细胞的非特异性抗体，特别是针对大量的胞内蛋白。
全长膜蛋白：具有天然构象的全长膜蛋白无疑是跨膜蛋白药物开发中的最佳抗原。然而全长膜蛋白在细胞中的表达水平通常比较低，而且不容易被分离纯化成为可溶性抗原。如何获得背景干净的富含靶点膜蛋白的可溶性抗原一直是抗体药研究的热点课题，也是缔码生物长期以来专注的研究方向。
为助力药物研发，解决跨膜蛋白靶点开发过程中抗原制备的难题，缔码生物专门搭建了多个跨膜蛋白全长表达平台。其中，纳米膜颗粒（MNP）平台是缔码生物主推的平台，它与病毒样颗粒（VLP）和外泌体（EXO）一起，是缔码现有的 3 大全长膜蛋白制备平台。同时缔码生物也正在开发去垢剂和合成纳米盘平台。
缔码生物全长膜蛋白表达方案
纳米膜颗粒（MNP）
使用缔码专有技术平台，通过系列理化手段在保证膜蛋白构象及活性的前提下直接提取高纯度的纳米级细胞膜颗粒。
代表 MNP 产品
Human CLDN6 full length protein-MNP
Human CCR8 full length protein-MNP
Human CD24 full length protein-MNP
Human CCR8 full length protein-MNP
Human CD24 full length protein-MNP
产品数据图
A. Negative Control 1: CCR8 full length membrane nanoparticles samples were stained only with Goat anti-human lgG 488 secondary antibody.
B. Negative Control 2: Control membrane nanoparticles samples were stained with anti-CCR8 antibody (BME100063) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
C. Negative Control 3: CCR8 full length membrane nanoparticles samples were stained with anti-Claudin 18.2 antibody （an irrelevant antibody) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
D. CCR8 full length membrane nanoparticles samples were stained with anti-CCR8 antibody (BME100063) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
B. Negative Control 2: Control membrane nanoparticles samples were stained with anti-CCR8 antibody (BME100063) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
C. Negative Control 3: CCR8 full length membrane nanoparticles samples were stained with anti-Claudin 18.2 antibody （an irrelevant antibody) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
D. CCR8 full length membrane nanoparticles samples were stained with anti-CCR8 antibody (BME100063) at 2 μg/mL, followed by Goat anti-human IgG 488 secondary antibody.
Western blot of CCR8 membrane nanoparticles (MNPs)
病毒样颗粒（VLP）
细胞在感染病毒后会从细胞膜表面通过一种类似细胞膜出芽的方式分泌 100-150nm 左右的纳米膜颗粒。VLP 带有完整的磷脂双分子层膜结构，膜蛋白以天然穿膜状态展示。
代表产品
Human Claudin-18.1 full length protein-VLP
Human Claudin-6 full length protein-VLP
Human CCR8 full length protein-VLP
Human CCR4 full length protein-VLP
Human CD24 full length protein-VLP
Human GPRC5D full length protein-VLP
Human Claudin-6 full length protein-VLP
Human CCR8 full length protein-VLP
Human CCR4 full length protein-VLP
Human CD24 full length protein-VLP
Human GPRC5D full length protein-VLP
产品数据图
ELISA plates were pre-coated with 0.5ug/per well purified human Claudin 18.2 VLP. Serial diluted Anti-Claudin18.2 monoclonal antibody (Zolbetuximab biosililar; IMAB362) solutions were added, washed, and incubated with secondary antibody before ELISA reading. From above data, the EC50 for IMAB362 binding with Claudin18.2 is 15.37ng/ml.
Negative Control 1: Claudin18.2 VLP samples were stained only with Goat anti-human IgG Fc-PE secondary antibody.
Negative Control 2: Control VLP samples were stained with anti-Claudin18.2 antibody（Zolbetuximab biosililar; IMAB362）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
Negative Control 3: Claudin18.2 VLP samples were stained with anti-BCMA antibody（an irrelevant antibody）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
Claudin18.2 VLP samples were stained with anti-Claudin18.2 antibody（Zolbetuximab biosililar; IMAB362）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
Negative Control 2: Control VLP samples were stained with anti-Claudin18.2 antibody（Zolbetuximab biosililar; IMAB362）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
Negative Control 3: Claudin18.2 VLP samples were stained with anti-BCMA antibody（an irrelevant antibody）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
Claudin18.2 VLP samples were stained with anti-Claudin18.2 antibody（Zolbetuximab biosililar; IMAB362）at 1ug/mL, followed by Goat anti-human IgG Fc-PE secondary antibody.
外泌体（EXO）
外泌体是直径在 30-150nm 的纳米颗粒，具有典型的脂质双分子层结构，主要来源于细胞膜内陷形成内体（endosome），然后内体内陷形成多囊泡体（MVBs），多囊泡体外膜与细胞膜融合后将外泌体释放到胞外基质中。外泌体膜携带了细胞膜蛋白，膜蛋白以天然穿膜状态展示。
代表产品
Human CD24 full length protein-EXO
Human GPRC5D full length protein-EXO
产品数据
Human CD24 full length protein-EXO
Human GPRC5D full length protein-EXO
产品数据
Figure 1. ELISA plates were pre-coated with 0.5 μg/per well purified human CD24 exosome. Serial diluted Anti-CD24 monoclonal antibody solutions were added, washed, and incubated with secondary antibody before ELISA reading. From above data, the EC50 is 69.61 ng/ml.
Figure 2. Nanoparticle Tracking Analysis of CD24 exosomes
Figure 3. TEM image of CD24 exosomes
去垢剂（Detergent）
去垢剂具有亲水极性基团和疏水非极性基团，能够使脂膜解体释放膜蛋白，并在溶液中为去膜状态下的膜蛋白提供疏水环境，维持和保护膜蛋白的疏水跨膜结构
代表产品
Claudin-6 全长膜蛋白（含 1%DDM 去垢剂）
Claudin-6 全长膜蛋白（含 1%DDM 去垢剂）
合成纳米盘（Synthetic Nanodisc）
与市面上大多数 MSP（膜支持蛋白）Nanodisc 不同，缔码生物研发的 Synthetic Nanodisc 能直接从完整的细胞中制作。在这个过程中，使用的合成高分子具有双重功能。首先，它溶解细胞膜，类似于洗涤剂，然后利用天然细胞磷脂在膜蛋白周围形成纳米盘结构。跨膜蛋白可整合到 Nanodiscs 中。
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