Optical control of protein-protein interaction has become a powerful technical means to manipulate and study various cellular processes. However, some basic processes, such as protein secretion, are usually difficult to manipulate. Creative BioMart has developed a light triggered protein secretion system based on UVR8 (a plant photoreceptor protein that forms a light unstable homodimer).
UVR8 fusion protein is conditionally isolated in the endoplasmic reticulum, and short light pulses can trigger its strong power to transport to the plasma membrane.
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UVR8 does not respond to excitation light used to image cyan, green or red fluorescent protein variants, allowing multicolor visualization of cell markers and secreted protein goods when it passes through cell secretion channels.
UVR8 has many unique characteristics, including the compositional formation of light unstable homodimer, slow reversal kinetics and UV-B absorption curve. It can perform polychromatic imaging of all widely used fluorescent proteins without activating photoreceptors.
Based on the system construction protocol we developed and optimized, our basic expression scheme is as follows
| Step | Description |
| Preparation of the plasmid | The expression vector driven by CMV promoter was constructed. On the vector, the coding of uvr8 is based on the C-terminal intracellular domain fused to vesicular stomatitis virus glycoprotein (VSVG). |
| Cell inoculation and transfection | HEK-293T cells will be adopted as the host, if there is no special need. The high-purity plasmid were transfected into host cells for incubation. |
| UVB light irradiation and sample collection | Samples were collected and stored 12-16 hours after the start of lighting |
As an alternative to HEK-293T cells, in principle, any other mammalian cells can be used as long as they can be transfected or transduced with the required genetic components. However, it may be necessary to adjust the protocol (e.g., transfection or transduction method, cell culture medium).
This technology can be widely used in many fields.
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