Difference between revisions of "Part:BBa K3765000"

Latest revision as of 11:23, 20 October 2021

SpyTag002

13 amino acid peptide ligand. Forms an isopeptide bond with SpyCatcher/SpyCatcher002 [1], used for anchoring two fusion proteins.

Usage and Biology

Streptococcus pyogenes has a fibronectin binding protein, FbaB. The collagen adhesin domain (CnaB2) of FbaB contains an internal isopeptide bond between Lys31 and Asp117 that confers exceptional stability. Zakeri et al. (PNAS, 2012) split the CnaB2 domain into an N-terminal protein fragment (SpyCatcher), containing Lys31, and a C-terminal peptide of 13 amino acids (SpyTag), containing Asp117. These parts spontaneously reconstitute to form an isopeptide bond under a wide range of pH values, temperatures, and redox conditions and in various detergents, remaining robust despite boiling or forces exceeding 1 nN, a breakage force at which streptavidin–biotin attachments and antibody–antigen interactions fail. This peptide tag can be used at the N- or C-termini of proteins or in the interior. In 2017, Keeble et al. (Angew. Chem. Int. Ed., 2017) reported SpyTag002 by using phage display and selecting for accelerated amidation and increased cross-reactivity with SpyCatcher. SpyTag002 is also 13 amino acid-long and has 4 residues that are different from SpyTag.

SpyTag reacts with SpyCatcher under a wide array of conditions, following second-order kinetics (Keeble et al., Angew. Chem. Int. Ed., 2017). Reaction was relatively independent of buffer salts, tolerating common non-ionic detergents and over 3 m urea. SpyCatcher002 reacted to 99 % completion with a small excess of SpyTag002-MBP. Conversely, SpyTag002-MBP reacted to 97 % completion with an excess of SpyCatcher002.

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]

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 <partinfo>BBa_K3765000 short</partinfo>
-aa peptide useful for protein tagging. Forms an isopeptide bond with SpyCatcher/SpyCatcher002, which can be used for protein pulldown/anchoring.
+amino acid peptide ligand. Forms an isopeptide bond with SpyCatcher/SpyCatcher002 [https://parts.igem.org/Part:BBa_K3765001], used for anchoring two fusion proteins.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
-<!-- -->
+''Streptococcus pyogenes'' has a fibronectin binding protein, FbaB. The collagen adhesin domain (CnaB2) of FbaB contains an internal isopeptide bond between Lys31 and Asp117 that confers exceptional stability. Zakeri et al. (PNAS, 2012) split the CnaB2 domain into an N-terminal protein fragment (SpyCatcher), containing Lys31, and a C-terminal peptide of 13 amino acids (SpyTag), containing Asp117. These parts spontaneously reconstitute to form an isopeptide bond under a wide range of pH values, temperatures, and redox conditions and in various detergents, remaining robust despite boiling or forces exceeding 1 nN, a breakage force at which streptavidin–biotin attachments and antibody–antigen interactions fail. This peptide tag can be used at the N- or C-termini of proteins or in the interior.  In 2017, Keeble et al. (''Angew. Chem. Int. Ed.'', 2017) reported SpyTag002 by using phage display and selecting for accelerated amidation and increased cross-reactivity with SpyCatcher. SpyTag002 is also 13 amino acid-long and has 4 residues that are different from SpyTag.
+SpyTag reacts with SpyCatcher under a wide array of conditions, following second-order kinetics (Keeble et al., ''Angew. Chem. Int. Ed.'', 2017). Reaction was relatively independent of buffer salts, tolerating common non-ionic detergents and over 3 m urea. SpyCatcher002 reacted to 99 % completion with a small excess of SpyTag002-MBP. Conversely, SpyTag002-MBP reacted to 97 % completion with an excess of SpyCatcher002.
+----
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K3765000 SequenceAndFeatures</partinfo>