Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E. coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.

丝绸是用途最广泛的生物材料之一，具有出色的机械强度和柔韧性等标志性特性。开发更环保的丝绸生产的一个潜在途径是利用蚕蛾 ( Bombyx mori ) 茧酶，这同时将增加使用副产品丝胶作为其他应用原料的可能性。Cocoonase 是一种丝氨酸蛋白酶，被蚕蛾用来软化茧，使其在完成变态后能够逃脱。Cocoonase选择性地降解茧的胶蛋白丝胶蛋白，而不影响由蛋白丝心蛋白制成的丝纤维。Cocoonase 可以在大肠杆菌中重组生产，然而，它仅作为不溶性包涵体被发现。为了解决这个问题并能够利用与基于大肠杆菌的表达系统相关的好处，我们开发了一种协议，可以生产毫克/升规模的可溶性和功能性蛋白酶。该协议的核心是在具有氧化还原电位的缓冲液中重新折叠蛋白质，该缓冲液针对天然和分子内二硫键的形成进行了优化。氧化还原电位与确定浓度的还原和氧化谷胱甘肽平衡。这个E 。 大肠杆菌基于生产协议，除了结构确定，还可以在催化功能和稳定性方面对椰子酶进行修饰。这些因素将成为开发替代丝绸生产方法的重要组成部分。