GGGG-Tag for Sortase-mediated Ligation x mCherry Fluorescence Protein

Profile

Name	GGGG-mCherry
Base pairs	1028
Molecular weight	28.5 kDa
Origin	synthetic, derived from Discosoma sp.
Parts	mCherry, GGGG-sequence, T7 Promoter, lac Operator, GASPAG Linker, Strep-Tag II, T7 Terminator
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. We use mCherry as an easily imaged reporter for checking if the coupling worked.
The coding sequence was cloned in pET24 vector, containing the sequence of mCherry, a GGGG-sequence, 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 T7 Terminator (BBa_K3187032), a Start-Codon (BBa_J70593) and a Stop-Codon (BBa_K2868029). The coding sequence consists of 851 bp which are translated to 260 amino acids.^[1]

Results

FRET-based assay to analyze Sortase ligation efficiency

Comparison of MGGGG-mCherry and GGGG-mCherry (BBa_K2868010

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 PstI site found at 854
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 854
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 915
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 854
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 854
1000
COMPATIBLE WITH RFC[1000]

[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]