Difference between revisions of "Part:BBa K5382130:Design"

Revision as of 13:12, 29 September 2024

CL7-linker-sfGFP_Green fluorescent protein and CL7 complex

Design Notes

In the expression of CL7-Linker-SFGFP, the detection and purification of its expression must be considered, otherwise the binding of CL7 and Im7 may be affected, thus affecting the membrane surface anchorings of the entire system, and the purity of sfGFP may be affected, thus affecting the subsequent dual fluorescence verification. This will affect the judgment of anchoring (film surface display system) validation. The double fluorescence verification here can be simply summarized as follows: The vesicle membrane was stained with Dil orange film dye, and CL7-linker-sfGFP was incubated with vesicles to anchor sfGFP on the membrane surface. After the samples were treated, the anchoring could be verified by the orange fluorescence of Dil and the green fluorescence of sfFGP under a confocal fluorescence microscope. After successful verification, it is proved that the membrane surface system is successfully constructed, and sfGFP can be changed into targeting elements such as single-chain antibodies and nano-antibodies to provide targeting.

Source

CL7 is a protein tag obtained by engineering CE7 to remove its DNA-binding and catalytic activity, preserving its high affinity binding ability to the corresponding inhibitory protein Im7. Linker is composed of Gly and Ser. Derived from wild-type GFP in the jellyfish Aequorea victoria, sfGFP has improved its folding properties through a series of mutations, allowing it to fold quickly and correctly and fluorescein brightly under a variety of environmental conditions. The source of composite parts is artificially constructed plasmid containing CL7 and sfGFP genes (PET23a-CL7-sfGFP).

Experimental results

We transferred pET23a-CL7-sfGFP into Escherichia coli BL21 and purified it to meet the use requirements. The results were as follows:

Figure 1. CL7-sfGFP protein debacteria-breaking supernatant was incubated with nickel column, imidazole eluent with gradient concentration above 80mM, and purified protein solution was concentrated and purified by ultrafiltration SDS-PAGE electrophoresis
Loading sequence: supernatant, precipitation, flow through, 20mM, 50mM, 80mM, 100mM, 150mM, 200mM, Marker, 300mM, 500mM

The protein size of CL7-sFGFP is 45KDa, which is consistent with the band about 45KDa in the protein glue
It can be seen that the purified protein concentration and purity are high, and subsequent incubation binding and fluorescence confocal experiments can be conducted to verify the binding of Inak-Im7 and CL7-sfGFP (see the engineer and result section of wiki for the results).

References