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Reagent to label proteins via lysine isopeptide bonds

Patent ·
OSTI ID:1998327
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Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction—first, cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate, and second, joining the terminal threonine to the nucleophilic lysine residue residing within the pilin motif of another pilin protomer. Informed by the high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and by developing structural variants of the sortase enzyme whose catalytic pocket has been unmasked by activating mutations, we have developed new reagents capable of forming isopeptide bonds in vitro. The reagents disclosed herein can catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile new platform for protein engineering and bio-conjugation that has major implications for biotechnology.
Research Organization:
Univ. of California, Los Angeles, CA (United States); Univ. of Texas, Austin, TX (United States)
Sponsoring Organization:
USDOE; National Institutes of Health (NIH)
DOE Contract Number:
FC02-02ER63421
Assignee:
The Regents of the University of California (Oakland, CA); The Board of Regents of The University of Texas System (Austin, TX)
Patent Number(s):
11,634,699
Application Number:
17/052,116
OSTI ID:
1998327
Country of Publication:
United States
Language:
English

References (9)

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds journal September 2009
Assembly of pili on the surface of Corynebacterium diphtheriae: Corynebacterium diphtheriae pili journal November 2003
Effects of Tonicity-Adjusting and Surfactant Agents on the Antimicrobial Activity of Alexidine journal March 2011
A Noncanonical Function of Sortase Enables Site‐Specific Conjugation of Small Molecules to Lysine Residues in Proteins journal October 2014
Sortase enzymes in Gram-positive bacteria: Sortase enzymes in Gram-positive bacteria journal November 2011
Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae journal June 2018
Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria journal July 2009
In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking journal May 2018
The “Lid” in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site journal May 2016

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