Fig 1.
A cartoon of the SpyCatcher/SpyTag-mediated cyclization of LUC.
We fused SpyCatcher and SpyTag to the N and C termini of LUC, respectively. An irreversible covalent bond formed between the reactive Lys of SpyCatcher and Asp of SpyTag during protein expression in vivo.
Fig 2.
Optimization of SpyCatcher protein: a double deletion at the N-terminus (26–47 aa) and C-terminus (134–138 aa) which named SpyCatcherΔNC.
All the internal regions of the SpyCatcherΔNC are identical with full-length SpyCatcher. Gray regions contain the His6-tag and tobacco etch virus (TEV) protease cleavage site. Blue regions indicate deleted parts in SpyCatcherΔNC.
Fig 3.
SDS-PAGE of the purified LUCs.
Lane M is the protein molecular weight markers. Lane 1, 2, 3, 4 are LUC, circular LUC, linear LUC and truncated circular LUC, respectively, purified using Ni-NTA affinity chromatography.
Fig 4.
The thermostabilities of the LUCs.
(A) The residual activities of the enzyme were measured after incubation at 45°C for different time intervals from 0 to 90 min (B) The residual activities of the enzyme were measured after incubation at various temperatures, from 25 to 55°C, for 15 min. Experiments were performed in triplicate and error bars correspond to the standard deviation.
Table 1.
Values are means ± S.D. for three independent measurements.
Fig 5.
The LUCs were incubated in various buffers, at the pH range of 2.0–12.0 for 30 min at 25°C and then were assayed for residual activity at room temperature. Experiments were performed in triplicate and error bars correspond to the standard deviation.
Fig 6.
Optimal pH values of the LUCs.
The activity was determined by injecting purified enzyme into assay buffers with different pHs (pH 2.0–12.0). Experiments were performed in triplicate and error bars correspond to the standard deviation.
Fig 7.
Optimal temperature of the LUCs.
The enzyme activity at a temperature gradient ranging from 10 to 60°C was determined. Experiments were performed in triplicate and error bars correspond to the standard deviation.
Fig 8.
Bioluminescence emission spectra (400–750 nm) produced by the LUCs at 25°C (A) pH 5.5 and (B) pH 7.8, respectively. Enzyme (50 μg) was assayed with 1.5 ml mix buffer containing 0.47 mM luciferin, 1.0 mM ATP, 10 mM MgSO4, 10 mM DTT and 25 mM tricine, adjusted to pH 7.8 and 5.5, respectively.
Fig 9.
CD spectra (190–260 nm) of linear and circular LUCs.
CD spectra were collected at 0.1 mg/mL LUC in 50 mM PBS (pH 7.8) at (A) 25°C and (B) 40°C, respectively.
Fig 10.
Fluorescence spectra of the LUCs.
Spectra were recorded at 25°C in 50 mM PBS (pH 7.8) using 20 μg/mL LUC solution. The excitation wavelength was 270 nm.