Difference between revisions of "Part:BBa K4040006"

Revision as of 01:31, 15 September 2021

TEV protease The TEV protease is a highly site-specific cysteine protease that is found in the Tobacco Etch Virus(TEV).The optimum recognition site for this enzyme is the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)(ENLYFQ(G/S)) and cleavage occurs between the Gln and Gly/Ser residues. The most commonly used sequence is ENLYFQG. The protease is used to cleave affinity tags from fusion proteins. The optimal temperature for cleavage is 30℃; also it can be used at temperatures as low as 4℃. It is recommended that the cleavage for each fusion protein be optimized by varying the amount of Recombinant Viral TEV Protease, reaction time, or incubation temperature. It can be removed by Ni2+ affinity resin. The usage in our project can be found in

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 431
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K4040006 short</partinfo>
-<h3><b>Structure of the spike(S) protein</b></h3>
+The TEV protease is a highly site-specific cysteine protease that is found in the Tobacco Etch Virus(TEV).The optimum recognition site for this enzyme is the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)(ENLYFQ(G/S)) and cleavage occurs between the Gln and Gly/Ser residues. The most commonly used sequence is ENLYFQG. The protease is used to cleave affinity tags from fusion proteins. The optimal temperature for cleavage is 30℃; also it can be used at temperatures as low as 4℃. It is recommended that the cleavage for each fusion protein be optimized by varying the amount of Recombinant Viral TEV Protease, reaction time, or incubation temperature. It can be removed by Ni2+ affinity resin.
-The spike(S) protein is a highly glycosylated protein embedded in the viral envelope of coronaviruses. There are three critical parts of the S protein: a &#947; carboxyglutamate-rich domain (Gla domain) in the amino terminus prepared for the binding with the phosphatidylserine located in the apoptotic cells or virus particles, four epidermal growth factor (EGF) like domains, and one sex hormone binding globulin (SHBG) domain which consists of two globular laminin G-like (LG) domains.
+The usage in our project can be found in
-[[File:SMMU-China-S protein.jpg|500px|thumb|center|]]
-<h3><b>The function of the S protein in SARS-CoV-2 infection</b></h3>
-The spike protein of the SARS-CoV-2 plays a crucial role in penetrating host cells and initiating infection. There are two function domains of the S protein, the S1 and the S2. The S1 domain has the specialized ability to bind with the ACE2 of the alveolar epithelial cells. After the S protein on the surface of SARS-CoV-2 binds with the extracellular domain of the ACE2 on the alveolar epithelial cells, the extracellular domain of the ACE will be lysed and the remaining SARS-CoV-2-ACE complex will be internalized with the assist of the Clathrin. After the fusion between the lipid bilayers of the SARS-CoV-2 and the alveolar epithelial cell, the RNA of the SARS-CoV-2 can finally be released and inserted into the targeted gene sequence to conduct virus replication.
-Considering the crucial role the S protein plays in the SARS-CoV-2 infection process, we are determined to construct a replication-defective pseudo- SARS-CoV-2 by expressing the S protein in the replication-defective vesicular stomatitis virus (Delta-G-VSV pseudovirus).
-The sequence of the spike protein is from GenBank (YP_009724390), and we ordered the DNA from a synthesis company.