Protein ladder

Polyacrylamide gel (12.5%) was used to perform sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Lee et al., 2020 (link)). The extracted myosin samples
(protein concentration, 0.5 mg/mL) were diluted twice (v/v) with sample
buffer (EBA-1051, Elpis Biotech, Daejeon, Korea) and heated at 95°C.
The samples (0.5 mg protein/mL) and protein ladder (3454A, Takara Bio,
Shiga, Japan) were loaded at 10 and 5 μL, respectively. The bands
were stained with a solution containing Coomassie brilliant blue, acetic
acid, and methanol overnight. Then, a buffer containing acetic acid and
methanol was used to de-stain the gels. The gels were scanned at an optical
resolution (63.5 μm/pixel) with a densitometer (GS-710, Bio-Rad
Laboratories) and analyzed using Image Master 2D Platinum v5.0 (GE
Healthcare, formerly Amersham Biosciences, Seoul, Korea).

All primers and plasmids used in this study are listed in Table1. E. coli DH10B (Promega, Beijing, China) was used for plasmid construction, promoter library construction, and characterization. E. coli BL21(DE3) was used for peptide expression. Plasmid pTrcHis2B (Invitrogen, Shanghai, People's Republic of China) harboring trc promoter and its mutated promoter library was constructed and preserved in our laboratory. PrimeStar DNA polymerase, restriction endonucleases, T4 DNA ligase, and protein ladder were purchased from Takara Biotechnology (Dalian, People's Republic of China). Plasmid DNA isolation and DNA gel purification were performed using AxyPrep Plasmid Miniprep and AxyPrep DNA gel extraction kits (Axygen Biosciences, Union City, USA). DNA primers, bovine serum albumin (BSA), and all other regents were provided by Sangon Biotech (Shanghai, People's Republic of China). The working concentration of ampicillin was 100 mg L⁻¹.