TEV Cleavage Site x GGGG-Tag for Sortase-mediated Ligation X mCherry Fluorescence Protein

Profile

Name	TVMV-GGGG-mCherry
Base pairs	1052
Molecular weight	29.4 kDa
Origin	synthetic, derived from Discosoma sp.
Parts	mCherry, GGGG-Sequence, TVMV site, T7 Promoter, lac Operator, RBS, araBAD promoter + RBS, GASPAG Linker, Strep-Tag II, Double Terminator for pDEStara2
Properties	Red fluorescent, Ex λ: 587nm, Em λ: 610 nm

Usage and Biology

mCherry (BBa_K3187026) is a red fluorescent protein. Which is a synthetic protein derived from Discosoma sp. by directed evolution. The N-terminal GGGG-sequence (BBa_K3187018) can be fused to a protein with a C-terminal LPETGG-Sortase A link (BBa_K3187019) by Sortase A. In order to remove the first methionine in front of the GGGG-Sequence a TVMV restriction site is cloned in the sequence. By removing the first methionine the linkage of LPETGG and GGGG-Sequences should work better. We use mCherry as an easily imaged reporter for checking if the coupling worked.
The coding sequence was cloned in pDEStara2 vector, containing the sequence of mCherry, a GGGG-sequence, a TVMV restriction site (BBa_K3187020), a GASPAG-Linker (BBa_K3187038), a Strep-Tag II (BBa_K3187025) for Purification, a T7 promoter with lac-operator and an RBS (BBa_K3187029), a T7TE terminator (BBa_K3187032), a Start-Codon (BBa_J70593) and a Stop-Codon (BBa_K2868029). Since pDEStara2 is a vector for dual expression it also contains an araBAD promoter with an RBS (BBa_K3187041) and a Double Terminator (BBa_K3187042). The coding sequence consists of 851 bp which are translated to 260 amino acids.^[1]

Methods

Purification

The GGGG-mCherry with TVMV Restrictionsite was heterologously expressed in E.coli BL21 and purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. The used affinity tag was Strep-Tag II. TVMV protease cleavage was performed over night at 4 °C.

Results

References

↑ Nathan Shaner, Robert Campbell, Paul Steinbach, Ben Giepmans, Amy Palmer and Roger Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology, 2004, 22: 1567-1572 [1]

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 324
Illegal PstI site found at 1133
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1197
Illegal PstI site found at 1133
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 239
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 324
Illegal PstI site found at 1133
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 324
Illegal PstI site found at 1133
Illegal AgeI site found at 74
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 56

[1] Nathan Shaner, Robert Campbell, Paul Steinbach, Ben Giepmans, Amy Palmer and Roger Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology, 2004, 22: 1567-1572 [1]