Difference between revisions of "Part:BBa K5036003"

Latest revision as of 13:14, 2 October 2024

TCS (Q, G)

Part Description

This is the TEV Cleavage Site which is a particular amino acid sequence that the TEV protease detects and cleaves

Usage

This is a particular segment of amino acids in the first chain of the dCas9(C)-TF-synVEGFR1 receptor can be cut by the TEV protease. This happens when VEGF attaches to our receptor and turns it on, releasing the dCas9(c) part

this figure illustrates the structure of TEV cleavage site (TCS(Q,G)) .

Dry lab Characterization

TCS is the site where TEV protease cleaves. It is a short sequence composed of 7 amino acids where anyone can apply a specific mutation to optimize the receptor’s response according to a near concentration needed. Similarly, it can act as an environmental sensor at which the receptor will be activated at a certain level so we measured the difference between the TCS and the mutant TCS in their binding stability with the TEV protease using Dynamut2 software tool

This figure confirmed that TCS(Q.L) carries a ΔG of -9.3 kcal mol-1 with the two domains of the TEV. While the TCS(Q.L) mutant form scored ΔG of -9.0 kcal mol-1 for (Q6G). On the other hand, TCS(Q.G) scored ΔG of -10.2 kcal mol-1 while its mutant form (E1D) scored ΔG of -10.1 kcal mol-1. .

Experimental Characterization

we digested dCas9(C)_NLS-Syn-VEGFR-1 grafetd with TCS (Q, G) and performed gel electrophoresis to detect the digested fragment.

This figure illustrates the digested dCas9(C)_NLS-Syn-VEGFR-1 prior to insert ligation .

We have done DNA gel electrophoresis to validate the cloning of our TCS (Q.G) into dCas9(C)_NLS-Syn-VEGFR-1 plasmid.

This figure illustrates the amplified fragments of our insert TCS (Q.G) within P3 .

Literature characterization

This research investigated how adding a TEV cleavage site impacted the production and solubility of nine specific mammalian proteins within bacterial cells. It's important to note that only these nine proteins were examined, and they are not representative of a broader selection. Four distinct tags were employed in the experiment: His-L, His-S, TEV1, and TEV2

This section details the amino acid sequences of four tags attached to the N-terminus of proteins. Each tag contains six histidine residues, highlighted for easy identification. Additionally, the TEV protease cleavage site (ENLYFQ/G) is bolded for the TEV1 and TEV2 tags. It's important to note that the His-S and TEV2 tags have an extra four amino acids (SAND) at the C-terminus, compared to the other two tags.

This section examines the analysis of expression and solubility of proteins with four different His-tags. The boxes indicate the expected size of the protein produced. (A) The mouse protein USF1 was produced at high levels from E. coli when the His-L, His-S, and TEV1 tags were present, but no protein was produced when the TEV2 tag was used. The protein remained dissolved when the His-L and His-S tags were used, but the protein formed clumps when the TEV1 tag was used. (B) The mouse latexin protein was produced at high levels with all four tags, but the protein only remained dissolved when the His-L or His-S tag was present. The presence of the TEV cleavage site (in TEV1 and TEV2 tags) resulted in insoluble proteins.

Reference

Kurz, M., Cowieson, N. P., Robin, G., Hume, D. A., Martin, J. L., Kobe, B., & Listwan, P. (2006). Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins. Protein expression and purification, 50(1), 68-73.‏

Sequence and Features