Sensing platform
A technology of sensors and biomolecules, applied in the field of sensors for detecting biological analytes, can solve unmet problems
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[0122] Preparation of GOx-PM-SWCNTs
[0123] 50mg SWCNT (rich in (7,6) of carbon nanotubes, Sigma Aldrich) was mixed with 50 mL of 2wt% sodium cholate (SC) (Sigma Aldrich) and sonicated at 1% in an ice bath using a tip sonicator (1 / 4 in. tip, Q Sonica Q700). Amplitude ultrasound. The SC-SWCNT suspension was ultracentrifuged at 164000 xg for 3 hours (Beckman Optima XPN-80). Approximately 80% of the supernatant was collected and stored at room temperature. 0.5mL of 25mg L -1 SC-SWCNT stock solution with 0.5 mL of approximately 3 mg mL -1 GOx-PM (or Gox) mix. The suspension was then dialyzed in 2L PBS (pH 7.4) in 14kDa MWCO dialysis tubing (D9777, SigmaAldrich) for 4 hours at 5°C. The dialyzed mixture was transferred to a 300 kDa MWCO dialysis apparatus (Spectra / Por Float-A-Lyzer, Spectrum Laboratories) and dialyzed against 2 L PBS buffer at 5°C for two days (during which the buffer was changed four times). GOx-PM-SWCNTs were stored at 4 °C.
[0124] GOx removal in...
Embodiment 1
[0130] Example 1: Mutagenesis of GOx
[0131] GOx variants with cysteine residues were designed that can be used to attach crosslinkers to specific surface regions.
[0132] To identify non-conserved surface-exposed amino acids, GOx homologues were analyzed and sequence conservation was mapped to the crystal structure of GOx from A. niger (PDB: 3QVP (Kommoju, P., et al. Probingoxygen activation) sites in two flavoprotein oxidases. Biochemistry 50, 5521-5534 (2011))).
[0133] From all residues amenable to mutagenesis to cysteine, four positions (D70, H446, A418 and K13) at different distances from the glucose-binding cavity of the enzyme were selected. figure 1A structural representation is shown showing amino acid residues K13, D70, A418 and H446 (blue) and the internal glucose-binding cavity with the FAD cofactor (red). Diagram showing the distance between the glucose-binding cavities above the si-face of the flavin ring (tricyclic isoalloxazine) (Kommoju, P.-R., et al...
Embodiment 2
[0135] Example 2: GOx expression
[0136] Wild-type GOx and K13C, D70C, A418C, H446C variants were expressed in optimized Pichia-producing yeast strains (Looser, V., et al. Cultivation strategies to enhance productivity of Pichiapastoris: A review. Biotechnology Advances. 33, 1177- 1193 (2014)), which contains a protein N-glycosylation pathway similar to that found in human cells (Brooks, S.A. Appropriate glycosylation ofrecombinant proteins for human use: Implications of choice of expressionsystem.Molecular Biotechnology.28, 241-256 (2004 )). Proteins were purified using size exclusion chromatography and stored in PBS (pH 7.0) supplemented with EDTA. Figure 4 The SDS-PAGE results shown in confirm the expected protein size (~85 kDa). Recombinant GOx from Pichia showed a slight increase in apparent molecules compared to the commercially available GOx from A. niger (about 80 kDa) (lane 1). This difference was attributed to a greater degree of glycosylation.
[0137] The e...
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