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Figure S1. Purification of BTGs (28 kDa). TGs from B. licheniformis (A) and B. subtilis (B) were purified by His-tag purification.

Figure S2. Confirmation of the BTG activities using MALDI-TOF. Q peptide (A) and hexalysine-BEBE (B) were used as substrates for BTG reaction. *A in (B) indicates chemical structure of Ac-hexalysine-BEBE. The crosslinked products, 2841.3 m/z (C) and 2841.3 m/z (D), were identified from B. licheniformis and B. subtilis reaction results, respectively.

Figure S3. Western blot of the crosslinked FPs. Lane 2 shows BTGs without His-tag. The bands of the dimeric FPs are weak. The TG substrates tags were attached at C-terminal region of each FP and the following His-tag was also located at the end of C-terminal region. By the crosslinking of two FPs, His-tag might be surrounded by the protein domains.

Figure S4. In-gel digestion of BTG reaction samples. A: The parts of higher molecular weight (40–48, 48–60, 60–80, and 80–100 kDa) than monomeric FPs were cut on SDS–PAGE gel. B: The underlined peptide sequences were recovered by the result of in-gel digestion.

Figure S5. In vivo color assay for BTG activity. A: Scheme of in vivo color assay using co-expression system. B: Selection of colonies showing relative low fluorescence intensities.

Figure S6. K subsite specificity of BLTGs. The Y-axis represents the frequency of a particular amino acid at a certain position, and the X-axis indicates the one-letter amino acid code. +: carboxy-group terminal to the glutamine (Q); −: amino-group terminal to the Q.

Figure S7. K subsite specificity of BSTGs. The Y-axis represents the frequency of a particular amino acid at a certain position, and the X-axis indicates the one-letter amino acid code. +: carboxy-group terminal to the glutamine (Q); −: amino-group terminal to the Q.

Figure S8. Comparison of fluorescence intensities for BTG reactions. A: The fluorescence intensities of BTG reactions were identified with various Q and K substrates. B: The explanation for each sample in A.

Table SI. Plasmid construction and primers. The restriction sites were underlined.

Table SII. Primers for tagged peptides in FP recombinants. The restriction site (XhoI) was underlined.

Table SIII. Measurement of fluorescence intensities with FPs. The FPs are abbreviated and listed in the table. The first character indicates a FP and the second stands for a tag. For example, CK means the eCFP tagged with hexa lysine. The fluorescence intensities of most FPs are not changed by TG activity but the decrease of yellow fluorescence intensity is remarkable.

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