Additional file 1 of Active secretion of a thermostable transglutaminase variant in Escherichia coli

Additional file 1: Table S1. Scripts used in this study. Table S2. mRNA folding energy based Top 10 and Bottom 10 variants. Table S3. Plasmids used in this study. Table S4. Synthetic double strand DNA fragment used in this study. Table S5. Primers used in this study. Figure S1. Expression of TGm1 in different forms. E. coli transformed with A pET-22b/pro-TGm1, B pET-22b/proH-TGm1, and C pET-22b-trxA-proH-TGm1. 1, 3, and 5: insoluble fractions; 2, 4, and 6: soluble fractions. E. coli was cultivated at 20 ℃ for 32 h after induction by adding IPTG at a final concentration of 0.1 mM when OD600 reached 1.0. Red arrow: A pro-TGm1, B proH-TGm1, and C TrxA-proH-TGm1. Figure S2. SDS-PAGE analysis of smTG variants purified by Nickel affinity chromatography. 1: FRAP-TGm1; 2: FRAPD-TGm1. Figure S3. Edman sequencing the N-terminus of recombinant TGm1 variants activated by dispase in vitro. pro-TGm1 and proH-TGm1 were intracellularly expressed in E. coli using pET-22b/pro-TGm1 and pET-22b/proH-TGm1, recombinantly expressed TGm1 variants were in vitro activated and purified by affinity chromatography using HisTrap (GE Healthcare, USA) column. Figure S4. E. coli carrying pETDuet/pelB-TrxA-proH-TGm1/TAMEP and pKJE7 was cultivated under 20 ℃ after induction by IPTG. Figure S5. MD simulation of FRAP-TGm1 and FRAPD-TGm1. A Hydrogen bonds within the N-terminus (FRAPDPDD) of FRAP-TGm1. B Hydrogen bonds within the N-terminus (FRAPDPDD) of FRAPD-TGm1. MD simulation was conducted for 100 ns using Gromacs-2020. C The modeled structure of FRAPD-TGm1. The N-terminal loop of FRAPD-TGm1 was colored in cyan. Figure S6. Sequence analysis of pro-TGm1 (GenBank ID: MZ516369). The number of nucleotide sequences (left side); Single underline: pro-region; Double underline: mature region of TGm1; Box, His-tag. Figure S7. Sequence analysis of TAMEP (GenBank ID: MZ516816). A start codon (ATG) was placed after the predicted signal peptide. Single underline: signal peptide [2]. Numbers (left side): the number of nucleotide sequences. Box, His-tag. For purification purpose, a His-tag was placed as shown in box while constructing the plasmid pETDuet/TAMEP; for co-expressing TAMEP with TGm1, the His-tag (as shown in box) was removed.