Part:BBa_K4766011

SpyTag-Streptavidin

The expression of SpyTag-Streptavidin protein

To attach the detection antibody to the protein cage, we employed Streptavidin (SA), using its interaction with biotin to link the antibody to the cage. For this purpose, we constructed the recombinant protein plasmid pET28a-SpyTag-Streptavidin (as shown in Figure 1).

Fig.1 Schematic design of the target protein gene sequence for the recombinant plasmid pET28a-SpyTag-Streptavidin.

Subsequently, E. coli was cultured at 22°C for 10 hours after adding 1mM of IPTG.

Fig.2 SDS-PAGE characterization results for SpyTag-Streptavidin

During our non-denaturing test of the sample, we identified an electrophoresis band at approximately 88kDa. However, in the denaturing gel electrophoresis, we not only observed this 88kDa band but also detected an additional band around 22kDa. As the boiling time increased, this 22kDa band intensified. The electrophoresis result is consistent with the tetramerization characteristics of SpyTag-Streptavidin. Based on the above observations, we preliminarily conclude that the protein we purified is SpyTag-Streptavidin. Optimization of expression conditions<\span> When SpyTag-Streptavidin is expressed at 22°C, we observed a substantial amount of inclusion bodies.

Fig.3 Solubility Analysis of SpyTag-Streptavidin.

Fig.4 Grayscale Analysis of SpyTag-Streptavidin Pellet and Supernatant under 22℃ Induction Conditions.

Data from grayscale analysis revealed that a substantial portion, nearly 80%, of the protein samples precipitated. This suggests the formation of a significant number of inclusion bodies during the SpyTag-Streptavidin purification process. We suspected that expressing SpyTag-Streptavidin at 22°C might cause rapid and erroneous protein folding, resulting in inclusion bodies. To address this, we lowered the expression temperature to 16°C.

Fig.5 Densitometry analysis of SpyTag-Streptavidin precipitate and supernatant.

Fig.6 Comparative densitometry analysis of SpyTag-Streptavidin precipitate and supernatant under induction conditions at 16°C and 22°C.

==The bioactivity of SpyTag-Streptavidin== To verify the bioactivity of SpyTag-Streptavidin, we took advantage of its specific binding to biotin. We added biotin-PEG-FITC to the protein sample to be tested for fluorescent characterization.

Table 1 Characterization results of SpyTag-Streptavidin-biotin-PEG-FITC.

Based on the data mentioned, while SpyTag-Streptavidin with Biotin-PEG-FITC did emit fluorescence, its intensity was almost negligible compared to that of SpyTag-Streptavidin and the blank control. Subsequently, we assembled SpyCatcher-Mi3 with SpyTag-Streptavidin at a 1:1 molar ratio, added Biotin-PEG-FITC, and characterized the fluorescence of FITC using NFC

Fig.7 a)The fluorescence representation of SpyCatcher-Mi3 : SpyTag-Streptavidin = 1:1. b)The fluorescence representation of SpyCatcher-Mi3 as control group

Analyzing the data obtained from NFC, the ratio of fluorescently labeled samples after assembly was significantly higher than that of unassembled samples, indicating the successful assembly of SpyCatcher-Mi3 with SpyTag-Streptavidin. Sequence and Features BBa_K4766011 SequenceAndFeatures