Developing an E. coli-Based Cell-Free Protein Synthesis System for Artificial Spidroin Production and Characterization
Journal
ACS Synthetic Biology
Journal Volume
14
Journal Issue
5
Start Page
1829
End Page
1842
ISSN
2161-5063
Date Issued
2025-04-21
Author(s)
Huang, Chang-Yen
Wang, Ruei-Chi
Hsu, Tzy-Shyuan
Hung, Tzu-Ning
Shen, Ming-Yan
Chang, Chung-Heng
Abstract
Spider silk spidroins, nature’s advanced polymers, have long hampered efficient in vitro production due to their considerable size, repetitive sequences, and aggregation-prone nature. This study harnesses the power of a cell-free protein synthesis (CFPS) system, presenting the first successful in vitro production and detailed characterization of recombinant spider silk major ampullate spidroins (MaSps) utilizing a reformulated and optimizedEscherichia coli based CFPS system. Through systematic optimization, including cell strain engineering via knockout generation, energy sources, crowding agents, and amino acid supplementation, we effectively addressed the specific challenges associated with recombinant spidroin biosynthesis, resulting in high yields of 0.61 mg/mL for MaSp1 (69 kDa) and 0.52 mg/mL for MaSp2 (73 kDa). The synthesized spidroins self-assembled into micelles, facilitating efficient purification compared to in vivo methods, and were further processed into prototype silk fiber products. The functional characterization demonstrated that the purified spidroins maintain essential natural properties, such as phase separation and fiber formation triggered by pH and ions. This tailored CFPS platform also facilitates versatile cosynthesis and serves as an accessible platform for studying the supramolecular coassembly and dynamic interactions among spidroins. This CFPS platform offers a viable alternative to conventional in vivo methods, facilitating innovative approaches for silk protein engineering and biomaterial development in a high-throughput, efficient manner.
Subjects
cell-free protein synthesis
fiber
phase separation
self-assembly
spider silk
spidroin
SDGs
Publisher
American Chemical Society (ACS)
Type
journal article