Pet30c

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.

The structural gene of wild-type GOx was prepared as previously described [7 (link)]. The gene was inserted into the multiple cloning site of the expression vector pET30c(+) (Merck KGaA, Darmstadt, Germany). Expression vectors of GOx mutants were prepared by site-directed mutagenesis using the QuikChange Mutagenesis Kit (Agilent Technology Inc., Santa Clara, CA, USA). The correctness of the mutations was confirmed using the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Expression and refolding of GOxs (both wild-type and mutant) were carried out as previously described [30 (link)].

All oligonucleotides were purchased from Eurofins Genomics (Luxembourg, Luxembourg). The sequences are indicated in the Supplementary Materials Table S1.
Phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), d(+)-glucose, sodium chloride, 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), glycerol, and Tween-20 were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). Kanamycin sulfate and d-desthiobiotin were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). A self-assembled monolayer (SAM) forming thiol reagent (dithiobis(succinimidyl undecanoate)) was purchased from Dojindo Laboratories Co., Ltd. (Kumamoto, Japan). LB broth, the bacterial host strain Escherichia coli BL21(DE3), and the expression vector pET30c(+) were purchased form Merck KGaA (Darmstadt, Germany). Anti-VEGF antibodies (MAB293 and BAF293) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Gold and platinum wires were purchased from TANAKA Kikinzoku (Tokyo, Japan). A silver/silver chloride (3M NaCl) reference electrode RE-1B (Ag/AgCl) was purchased from BAS Inc. (Tokyo, Japan).