Peptide ligase and use thereof
A peptide tag, isopeptide bond technology, applied in the field of peptide ligase and its application, can solve the problems of low activity, slow reaction rate, low conjugation yield and the like
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Embodiment 1
[0209] Example 1 - Development of Peptide Ligase (SnoopLigase) and Peptide Tag
[0210] RrgA (SEQ ID NO: 4) is an adhesin from Streptococcus pneumoniae, a Gram-positive bacterium that causes septicemia, pneumonia, and meningitis in humans. A spontaneous isopeptide bond is formed in the D4 immunoglobulin-like domain of RrgA between residues Lys742 and Asn854.
[0211] The inventors "divided" the D4 domain into three parts, a pair of peptide tags called SnoopTag (residues 734-745 of RrgA, SEQ ID NO: 9) and RrgATag2 (residues 838-860 of RrgA, SEQ ID NO: 10), and the protein known as RrgA Ligase (RrgALigase) (residues 743-846, SEQ ID NO: 8). Notably, there is a 9 amino acid overlap between the N-terminus of RrgA tag 2 and the C-terminus of RrgA ligase. Likewise, there is a 3 amino acid overlap between the C-terminus of the peptide tag and the N-terminus of the RrgA ligase. In addition, the RrgA ligase and RrgA tag 2 sequences incorporate modifications relative to the native R...
Embodiment 2
[0221] Example 2 - Exploring Ligase-Mediated Peptide-Peptide Ligation
[0222] To verify the proposed reaction mechanism and the specificity of the probe ligase residue linkage, probe tags Jr and Dog tags were fused to model proteins. The Dog tag was fused to a Small Ubiquitin-like Modifier (SUMO), while the probe tag Jr was fused to an Affimer for HER2. Mixing of SUMO-Dog tag and SnoopTag Jr-AffiHER2 (SnoopTagJr-AffiHER2) with Snoop ligase resulted in the appearance of a new higher molecular weight band representing the covalently linked ligation product. This band has the expected molecular weight and is resistant to boiling in SDS loading buffer. Mutation of any of the three reactive triplet residues (lysine at position 9 in Jr of the Probe tag, asparagine at position 17 in the Dog tag, and glutamic acid at position 61 in the ligase of Probe) Mutations prevented the appearance of ligation product bands ( figure 2 B). Mass spectrometry gave the expected molecular weig...
Embodiment 3
[0223] Embodiment 3-probe ligase reaction conditions
[0224] The probe ligase reaction works well around neutral pH with little difference from 7.25 to 8.75 ( Figure 4 A). Effective connection ( Figure 4 B). Although the reaction is most efficiently performed using Tris borate buffer in the absence of NaCl, the probe ligase is functional in the presence of extracellular concentrations of NaCl ( Figure 5 A). Probe ligase reacted well in the presence of common detergents Tween 20 and Triton X-100, up to 2%, but SDS inhibited the reaction ( Figure 5 B). Adding the protein stabilizer glycerol at 15-30% (v / v) can increase the reaction rate ( Figure 5 C).
[0225] The melting temperature of the probe ligase obtained from DSC was 45°C, and the full activity was restored after heat treatment up to 70°C. Some activity was recovered after heating at 99°C.
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