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Simple production of resilin-like protein hydrogels using the Brevibacillus secretory expression system and column-free purification
Authors:Toshimasa Homma  Shu Terui  Fuki Yokoyama  Saki Okino  Sora Ohta  Chihiro Kato  Naoki Haraguchi  Ikuhide Fujisawa  Shinichi Itsuno  Lily Zuin Ping Ang
Institution:1. Division of Chemical Engineering and Biotechnology, National Institute of Technology, Ichinoseki College, Ichinoseki Iwate, Japan;2. Department of Applied Chemistry & Life Science, Toyohashi University of Technology, Toyohashi, Aichi, Japan;3. Department of Applied Chemistry & Life Science, Toyohashi University of Technology, Toyohashi, Aichi, Japan

National Institute of Technology, Gifu College, Motosu, Gifu, Japan

Abstract:Resilin, an insect structural protein, has excellent flexibility, photocrosslinking properties, and temperature responsiveness. Recombinant resilin-like proteins (RLPs) can be fabricated into three-dimensional (3D) structures for use as cell culture substrates and highly elastic materials. A simplified, high-yielding production process for RLPs is required for their widespread application. This study proposes a simple production process combining extracellular expression using Brevibacillus choshinensis (B. choshinensis) and rapid column-free purification. Extracellular production was tested using four representative signal peptides; B. choshinensis was found to efficiently secrete Rec1, an RLP derived from Drosophila melanogaster, regardless of the type of signal peptide. However, it was suggested that Rec1 is altered by an increase in the pH of the culture medium associated with prolonged incubation. Production in a jar fermentor with controllable pH yielded 530 mg Rec1 per liter of culture medium, which is superior to productivity using other hosts. The secreted Rec1 was purified from the culture supernatant via (NH4)2SO4 and ethanol precipitations, and the purified Rec1 was applied to ring-shaped 3D hydrogels. These results indicate that the combination of secretory production using B. choshinensis and column-free purification can accelerate the further application of RLPs.
Keywords:Brevibacillus choshinensis  column-free purification  extracellular expression  photocrosslinking  resilin-like proteins  three-dimensional hydrogel
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