Difference between revisions of "Part:BBa K3765012"

Latest revision as of 17:22, 20 October 2021

PelB_Signal+10xHis+SpyCatcher002+dCBD

CDS of fusion protein consisting of SpyCatcher002 linked to dCBD (double cellulose binding domain), which can be used for anchoring SpyTagged proteins onto a microcrystalline cellulose (MCC) matrix.

Usage and Biology

Contains a PelB signal peptide and 10xHis Tag attached at the N terminus, for extracellular secretion and purification purposes respectively. A pair of AgeI sites have been added on either side of the SpyCatcher002 module for it to be easily removed in case any other anchoring strategy needs to be employed. The AgeI site after digestion followed by ligation also provides a site of linearization for a plasmid carrying this insert, so that virtually any module can be cloned upstream of the dCBD by Gibson Assembly, InFusion Cloning etc.

Fig 1. Feature Map

A tertiary structure for this fusion protein was predicted using AlphaFold. The SpyCatcher002 and dCBD modules fold independently due to the presence of a flexible 29 amino acid compound linker (GSSGS + 24 amino acid N terminal linker from dCBD) separating them. The Lys residue in the SpyCatcher002 which is responsible for forming an isopeptide bond with an Asp residue in SpyTag002 is exposed on the surface, and this binding is not hampered by the presence of the dCBD module.

Fig 2. Predicted cartoon model [N- to C- direction is blue to red]

This fusion protein is also appreciably soluble in physiological conditions, and can be purified by Ni-NTA affinity chromatography.

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 634
Illegal XhoI site found at 276
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 106
Illegal AgeI site found at 472
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 361

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
 Contains a PelB signal peptide and 10xHis Tag attached at the N terminus, for extracellular secretion and purification purposes respectively. A pair of AgeI sites have been added on either side of the SpyCatcher002 module for it to be easily removed in case any other anchoring strategy needs to be employed. The AgeI site after digestion followed by ligation also provides a site of linearization for a plasmid carrying this insert, so that virtually any module can be cloned upstream of the dCBD by Gibson Assembly, InFusion Cloning etc.
-[[File:T--IISc-Bangalore--images--Map_C1.png|thumb|center|Fig 1. Feature Map]]
+[[File:T--IISc-Bangalore--images--Map_C1.png|thumb|center|900px|Fig 1. Feature Map]]
 A tertiary structure for this fusion protein was predicted using [https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb AlphaFold]. The SpyCatcher002 and dCBD modules fold independently due to the presence of a flexible 29 amino acid compound linker (GSSGS + 24 amino acid N terminal linker from dCBD) separating them. The Lys residue in the SpyCatcher002 which is responsible for forming an isopeptide bond with an Asp residue in SpyTag002 is exposed on the surface, and this binding is not hampered by the presence of the dCBD module.