title: Biogenesis of silk fibroin (Bombyx mori) production in bacteria and baculovirus-infected insect cell system
Creators: name: Butiman, Chirapha
Publisher: name: University of Glasgow
Year: 2023
Subjects: subject: fibroin, fibroin heavy chain (H-chain), fibroin light chain (FIBL), fibrohexamerin (P25).

description: This research project has established three main hypotheses: 1) bacterial expression systems be used to produce three of the core silk proteins of Bombyx mori (P25 glycoprotein, FIBL, and Hchain); 2) baculovirus-insect cell expression systems be used to produce three of the core silk proteins (P25 glycoprotein, FIBL, and H-chain); and 3) H-chain interacts independently from the molecules of silk proteins and other proteins because of its superior properties, such as high self-assembly and adhesive formation. The protein production in the bacterial system of the two lower molecular fibroin proteins (P25 and FIBL, MWs about 30 kDa) was first carried out by labelling with His6x-tagged at the C-terminus, cloned into pET28a, and expressed in two groups of bacterial strains: Group 1 (unenhanced disulfide bond formation), including three bacterial cells; BL21 (DE3), C41 (DE3), RosettaTM (DE3)pLysS (Novagen); and Group 2 (enhanced disulfide bond formation), containing one bacteria strain, SHuffle® T7. The findings revealed that FIBL protein was expressed as inclusion bodies (IBs), which needed to be solubilised using 7M urea to recover the active protein. FIBL protein production was performed using a larger volume of (250mL) LB media with the optimal growth condition of 0.5mM IPTG and induced overnight at 25ºC. The FIBL obtained the secondary structure using circular dichroism. However, the P25 protein expression had the most potential growth in SHuffle® T7 of 0.5mM IPTG and was induced overnight at 25ºC, but its construct was not stable after maintenance at -20ºC for about three months. In the second attempt, the P25 glycoprotein was labelled on both sides with His6x-tagged + 3C protease (N-terminus), whereas TEV protease + His6x-tagged was added (C-terminus) to the pFBDM plasmid and expressed in Sf21 cells (Spodoptera frugiperda). The findings showed the system can promote the complex N-linked P25 glycoprotein. The P25 protein expression showed two forms of glycosylation in the Sf21 cells: nonsecretion (semi-glycosylated) and secretion (medium fractions/glycosylated). The semi-glycosylated P25 contained β-pleated sheet (33.4%), un-ordered (31.5%), turns (20.8%), and helix (14.3%), respectively. In contrast, P25 (secreted) consisted of a βpleated sheet (47.70%), un-ordered (35.05%), turns (17.05%), and helix (0.20%), respectively. This is also the first finding about the P25 glycoprotein secondary structure. The H-chain is a large subunit (about 350 kDa) with a high crystallinity structure that cannot be easily expressed using the classical cloning technique. Therefore, the better choice is to isolate the Hchain from the native solid posterior silk gland (PSG), B. mori. The native H-chain fibroin was satisfactorily isolated from the solid fibroin prepared from the cocoon shells by the first purification with gel filtration chromatography and the second purification using ion exchange chromatography. Three fibroin (H-chain, FIBL, and P25) element interactions and formations among fibroin molecules revealed the assembled relationship as a stoichiometry complex (H(6):FIBL(6):P25(1)), and other complexes were investigated by a pull-down assay. Additionally, the H-chain was employed to form multimers and interactions that have potential with selected human proteins, CLIC1 and CLIC4, in heterodimeric form, a promising finding for novel medical applications. It was interesting that the native H-chain alone always forms homodimers around 720 kDa.