Difference between revisions of "Part:BBa K1319004"

Revision as of 01:51, 17 October 2014

TEV protease with anti-self cleavage mutation S219V, codon optimized for E. coli

This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.

The TEV Protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part K1319016.

ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.

The TEV protease is commonly used as a biochemical tool to cleave affinity tags from purified proteins like His-Tags. The high specifity makes the protease relatively non-toxic in vitro and in vivo. The molecular weight of the TEV protease is 27 kDa.

Usage and Biology

The TEV Protease was used and characterizes in the K1319008 construct.

To characterize the TEV protease we used the fusion protein K1319014. This fusion protein contains GFP (E0040) bound to a dark quencher (REACh2/K1319002) over a linker which includes the TEV protease cleavage site. If the TEV protease successfully cuts the linker, GFP and its quencher would separate and the FRET (Förster Resonance Energy Transfer) system would be shut down. This would result in an increased GFP fluorescence.

To demonstrate this behaviour a double plasmid system was designed using the biobrick K1319013 in a pSB3K3 backbone and K1319008 in a pSB1C3 backbone. Also I20260 was used as a positive control because it produces the same GFP as used in the fusion protein and is regulated by the same promoter, RBS and Terminator on the same plasmid backbone. B0015 was used as negative control.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 13: / Line 13: @@
 The TEV Protease was used and characterizes in the [https://parts.igem.org/Part:BBa_K1319008 K1319008] construct.
+To characterize the TEV protease we used the fusion protein [https://parts.igem.org/Part:BBa_K1319014 K1319014]. This fusion protein contains GFP ([https://parts.igem.org/Part:BBa_E0040 E0040]) bound to a dark quencher ([https://parts.igem.org/Part:BBa_K1319002 REACh2/K1319002]) over a [https://parts.igem.org/Part:BBa_K1319016 linker] which includes the TEV protease cleavage site. If the TEV protease successfully cuts the linker, GFP and its quencher would separate and the FRET (Förster Resonance Energy Transfer) system would be shut down. This would result in an increased GFP fluorescence.
+To demonstrate this behaviour a double plasmid system was designed using the biobrick K1319013 in a pSB3K3 backbone and K1319008 in a pSB1C3 backbone. Also [https://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because it produces the same GFP as used in the fusion protein and is regulated by the same promoter, RBS and Terminator on the same plasmid backbone. [https://parts.igem.org/Part:BBa_B0015 B0015] was used as negative control.