MOESM1 of Extracellular production of the engineered thermostable protease pernisine from Aeropyrum pernix K1 in Streptomyces rimosus
Creators: name: Šnajder, Marko
name: Rincón, Andrés Carrillo
name: Magdevska, Vasilka
name: Bahun, Miha
name: Kranjc, Luka
name: Paš, Maja
name: Juntes, Polona
name: Petković, Hrvoje
name: Nataša Poklar Ulrih
Publisher: name: figshare
Year: 2019
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Evolutionary Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: Additional file 1: Table S1. Codon distribution of the wild-type pernisine sequence from A. pernix and the codon-optimised pernisine sequence from S. rimosus. Figure S1. Tandem mass spectrometry (MS/MS) analysis of the N-termini and C-termini of the codon-optimised prepropernisine (A) and the codon-optimised processed pernisine (B). Individual identified peptides are aligned with the pernisine sequence. Figure S2. Model of the three-dimensional structure of propernisine (A) and processed pernisine (B), with the Ca2+ binding sites indicated (C). Note that the first 31 amino acids are not shown in the model structure of pernisine. Propernisine consists of the proregion (V31-A92; turquois), the catalytic triad (D149, H184, S355), and the Ca2+ ions (green spheres). Processed pernisine form (Q93-V430). Frame C presents four selected Ca2+ binding sites that interact with either amino acids or water (H2O). Figure S3. Production of codon-optimised srT-pernisine (from plasmid construct pVFPER5) in total cell lysates from S. rimosus grown in different media. TSB, tryptic soy broth; MM, minimal medium; G3, G4, G5, complex production media at 3, 4 and 5 days. The protein was transferred onto nitrocellulose membranes as dot blots, and His6-tagged pernisine was detected using anti-His5 antibodies. Figure S4. Amino-acid alignment of pernisine and Tk-subtilisin with the Ca2+ binding sites indicated. Yellow, identical amino acids; green, related amino acids. Known Ca2+ binding sites from Tk-subtilisin are marked with coloured asterisks. Site 1, blue; site 2, red; site 3, black; site 4, green; site 5, violet; site 6, orange; site 7, grey. Figure S5. Comparison of the nucleotide sequences of prepropernisineWT and propernisineCO. The predicted amino-acid sequence of the prepropernisine is indicated in the single-letter amino-acid code. Differences in the DNA sequences are shown in bold letters. Single underlined, amino acids of the signal sequence; double underlined, amino acids of the predicted proregion; remaining amino acids, active pernisine. The amino acids involved in the predicted catalytic triad are in yellow, as Asp149, His184 and Ser355. The start codon is marked with an asterisk (*); the stop codon with a hyphen (−). Note that only ATG was used as the start codon for the heterologous expression.