Part:BBa_K2683009
Arc minimal protein construct derived from HIV homology
The Arc protein is originally found in mammalian brain tissue. It is known to form capsid structures that can delivery RNA from one cell to the other; specifically neural cells[1]. The arc minimal construct is based on the arc protein sequence that has similar homologies to the HIV sequence. This Novel protein that we have created is hypothesized to retain its ability to form capsids and deliver RNA. This part has been Codon optimized for expression in E.coli .
In order to highly characterize Arc minimal, we have used a series of experiments including protein purification, Transmission Electron Microscopy and analytical Ultracentrifugation.
Figure 1- A 15% SDS-PAGE gel showing the purification of Arc minimal. The lysate of the sample was run through a sucrose cushion, dialyzed against 1XPBS and then run on a size exclusion column. The protein is seen at the 40kDa range, indicating that it is likely dimerizing as the expected size is at 20kDa.
Figure 2 - This is a chromatogram that shows the size exclusion chromatography purification of arc min. Elutions based on the peaks were taken and run on a gel and the rest of the samples were used for further testing.
Figure 3- Transmission Electron Microscopy image of Arc minimal proteins. The size of the capsid is about 30nm and was able to form spontaneously without the presence of RNA. Therefore, the hypothesized ability to form capsid structures is proven to occur.
Figure 4- Analytical ultracentrifugation sedimentation results and simulations. (top left) Converted relative light intensity sedimentation rates into absorbance to compare to simulations. (top right) AUC sedimentation rates of Arc-min (top-right) measured in intensity of light at 217 nm and spun at 30,000 rpm. (bottom left) Modeled expected sedimentation rates of an Arc-min monomer and (bottom right) hexamer. Simulated data is based on estimated molar masses and globular anisotropies, using identical experimental conditions used in the actual experiment.
Sequence and Features Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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