School of Physical Science and Technology, ShanghaiTech University, Shanghai, China.
School of Materials Science and Engineering, Zhengzhou University, Zhengzhou, China.
Methods Mol Biol. 2021;2347:83-96. doi: 10.1007/978-1-0716-1574-4_8.
Collagen represents the major structural protein of the extracellular matrix. The desired mechanical and biological performances of collagen that have led to its broad applications as a building block in a great deal of fields, such as tissue engineering, drug delivery, and nanodevices. The most direct way to obtain collagen is to separate and extract it from biological tissues, but these top-down methods are usually cumbersome, and the structure of collagen is usually destroyed during the preparation process. Moreover, there is currently no effective method to separate some scarce collagens (such as collagen from human beings). Alternatively, bottom-up assembly methods have been developed to obtain collagen assembly or their analogs. The collagen used in this type of method is usually obtained by genetic recombination. A distinct advantage of gene recombination is that the sequence structure of collagen can be directly customized, so its assembly mode can be regulated at the primary structure level, and then a collagen assembly with a predesigned configuration can be achieved. Additionally, insights into the assembly behavior of these specific structures provide a rational approach to understand the pathogenic mechanisms of collagen-associated diseases, such as diabetes. In this chapter, Type I collagen is used as an example to introduce the key methods and procedures of collagen recombination, and on this basis, we will introduce in detail the experimental protocols for further assembly of these recombinant proteins to specific structures, such as fibril.
胶原代表细胞外基质的主要结构蛋白。胶原具有理想的机械和生物学性能，使其广泛应用于组织工程、药物输送和纳米器件等多个领域作为构建模块。获得胶原的最直接方法是从生物组织中分离和提取，但这些自上而下的方法通常很繁琐，而且胶原的结构在制备过程中通常会被破坏。此外，目前还没有有效的方法来分离一些稀缺的胶原（如人类胶原）。另一种方法是自下而上的组装方法，以获得胶原组装或其类似物。此类方法中使用的胶原通常通过基因重组获得。基因重组的一个明显优势是可以直接定制胶原的序列结构，因此可以在一级结构水平上调节其组装模式，从而实现具有预定结构的胶原组装。此外，对这些特定结构的组装行为的深入了解为理解与胶原相关的疾病（如糖尿病）的发病机制提供了一种合理的方法。本章以 I 型胶原为例，介绍了胶原重组的关键方法和步骤，并在此基础上详细介绍了进一步将这些重组蛋白组装成特定结构（如纤维）的实验方案。
