Construction of CYTc-SpyCatcher and ST-Enzyme Fusion Proteins

The amino acid sequences of the CYTc and SpyCatcher have been previously described [43 (link),50 (link)]. To fuse the SpyCatcher to the C-terminus of CYTc, the following genes were synthesizedo: the structural gene of CYTc (residues 16–130) without its original signal sequence with an NcoI-pelB signal sequence HisTag at the 5′ end, and the structural gene of SpyCatcher (residues 21 to 104) with HindIII at the 3′ end was subsequently coded. This gene fragment, NcoI-pelB-HisTag-CYTc-SpyCatcher-HindIII, was amplified by a polymerase chain reaction (PCR) and inserted into pET30c(+) (Merck KGaA, Darmstadt, Germany) to construct an expression vector of the CYTc-SpyCatcher. To construct the ST-Enzyme expression vector, the structural gene of the SpyTag was added at the 5′ side of the structural gene of the FAD-dependent GDH derived from Aspergillus flavus [44 (link),45 (link)], a G52V mutant of DAAOx derived from Rhodotorula gracilis [18 (link),46 (link)], and an A96L/N212K double mutant of LOx derived from Aerococcus viridans [15 (link),47 (link)] by PCR amplification. Each gene fragment coding, ST-GDH, ST-DAAOx, or ST-LOx was inserted to the NdeI-HindIII site of pET28a(+) (Merck KGaA, Darmstadt, Germany). The N-terminus of each ST-Enzyme contained a His-Tag derived from the vector. All the constructed expression vector information is shown in Supplementary Materials Figure S1.