Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof

An isopeptide bond and identity technology, applied in the field of two-part joints, can solve the problems of limited reaction rate and suboptimal reaction rate

Pending Publication Date: 2020-01-17
OXFORD UNIV INNOVATION LTD
View PDF9 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this regard, it is hypothesized that the rate at which the spy-tag peptide and the spy-capture peptide interact may limit the rate of the reaction
Therefore, the development of a suitable screening system requires the selection of reaction conditions under which the reaction rate between the spy tag peptide and the spy capture polypeptide is not optimal

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof
  • Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof
  • Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0273] Example 1 - Spy Tag (SEQ ID NO:6) Phage display optimization

[0274] Spy tags / spy captures are an unconventional approach to peptide interactions, and some features of the interactions cannot be predicted by rational design. Selection from phage libraries has been around for decades, and the difficulty has generally been the detection of weak interactions rather than the challenge of screening for irreversible interactions. We initially established model selection to study efficient isopeptide bond formation selection.

[0275] We found that the first key function to be able to successfully pan the spy tag - phage is to capture the spy capture agent (SEQ ID NO: 7) bait in the capture solution, rather than attach the spy capture agent to the beads. Solution capture reagents allow for easy titration of titrant concentrations and reduce the background of non-specific binding of phage to beads ( figure 1 ).

[0276] The second key feature is the specific elution o...

Embodiment 2

[0280] Embodiment 2-spy capture agent (SEQ ID NO:7) Phage display optimization

[0281] Phage display selection of spy capture agents is performed in a manner similar to selection of spy tag variants, although displaying split proteins on the phage surface provides a further challenge. The key features we found to be important for efficient selection are the TEV protease cleavage site between the spy capture agent and pIII on the phage (allowing specific elution of the phage from the beads) and co-translational translocation using the DsbA signal sequence, thereby Improved display of Spy Capture Agents on pIII. The bait was biotinylated Avi-tag-Spy-MBP, and the spy-capture variants were prepared by error-prone PCR ( Figure 4 A). We initially optimized model selection using the desired bait (spy tag) or negative control spy tag DA, which non-covalently binds to the spy capture agent but does not react. Recovered phage were evaluated by qPCR. This selection showed tha...

Embodiment 3

[0285] Embodiment 3-Verification of mutation rate of spy label 002 and spy capture agent 002

[0286] After having Spy Tag 002 and Spy Capture Agent 002, we carefully verified their response behavior. We confirmed the putative key role of reactive residues by showing the response to ablation of a single mutation in spy tag 002 (DA) or spy capture agent 002 (EQ) ( Figure 9 A).

[0287] The spy tag / spy capture agent reaction is effective at high concentrations. In order to analyze the reaction at low concentrations, after polyacrylamide electrophoresis, it was reacted with superfolded GFP (superfolder GFP, sfGFP) for fluorescence detection of the formation of covalent bonds. If the sample is not boiled, sfGFP can remain folded and fluoresce even in the presence of SDS. This analysis showed that the reaction rates of SpyTag 002 and Spy Capture Agent 002 were significantly increased compared to the parental version ( Figure 9 B). As expected, the difference was smaller wh...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention relates to a two-part linker comprising a peptide tag (peptide) and a polypeptide (protein) that is capable of spontaneously forming an isopeptide bond, particularly wherein: a)said peptide comprises an amino acid sequence as set forth in SEQ ID NO: 1, wherein: (i) X at position 1 is arginine or no amino acid; (ii) X at position 2 is glycine or no amino acid; (iii) X at position 5 is histidine or threonine; (iv) X at position 11 is alanine, glycine or valine; and (v) X at position 14 is arginine or lysine, wherein when X at position 1 is no amino acid, X at position 2 isno amino acid; and b) said polypeptide comprises: i) an amino acid sequence as set forth in SEQ ID NO: 2; ii) a portion of (i) comprising an amino acid sequence as set forth in SEQ ID NO: 101; iii) an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 2, wherein said amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80and one or more of the following: 1) threonine at position 5; 2) proline at position 16; 3) arginine at position 40; 4) histidine at position 65; 5) proline at position 92; 6) aspartic acid at position 100: 7) glutamic acid at position 108; and 8) threonine at position 116, wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 2; or iv) a portion of(iii) comprising an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 101, wherein the amino acid sequence comprises a lysine at position 10, a glutamicacid at position 56 and one or more of the following: 1) arginine at position 16; 2) histidine at position 41; 3) proline at position 68; and 4) aspartic acid at position 76, wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 101, and wherein said peptide and polypeptide are capable of spontaneously forming an isopeptide bond between the aspartic acid residue at position 10 of SEQ ID NO: 1 and the lysine residue at position 34 of SEQ ID NO: 2 or position 10 of SEQ ID NO: 101.

Description

technical field [0001] The present invention relates to a two-part linker comprising a peptide tag and a polypeptide (protein) capable of spontaneously forming isopeptide bonds. In particular, the two-part linkers of the invention can be viewed as pairs of peptide tag and polypeptide binding partner homologues that, when contacted under conditions that allow the spontaneous formation of isopeptide bonds between the peptide tag and its polypeptide binding partner, Pairs can be conjugated via covalent bonds. Also provided are nucleic acid molecules encoding said each part of said two-part linker (ie, peptide tag and polypeptide binding partner), vectors comprising said nucleic acid molecules, and host cells comprising said vectors and nucleic acid molecules. Kits comprising said two-part linker (ie peptide tag and polypeptide binding partner) and / or nucleic acid molecule / carrier are also provided. Also provided are methods of producing said two-part linkers (ie peptide tag and...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/315
CPCC07K14/315C07K2319/02C07K2319/20C07K2319/21C07K2319/24C07K2319/50
Inventor M·豪沃思A·基布尔
Owner OXFORD UNIV INNOVATION LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap