ArticleJournal of the American Chemical Society2009
Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase.
Article in Journal of the American Chemical Society, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 39 papers.
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Who cites it
39 citing papers in PubMed, 107 citations in OpenAlex.
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- Library-on-Library Intercellular Labeling for Selection of Biotin Ligase and Acceptor Peptides.Chembiochem : a European journal of chemical biology · 2026Article
- Homogeneous antibody-drug conjugates with dual payloads: potential, methods and considerations.mAbs · 2025Review
- Utilizing small molecules to probe and harness the proteome by pooled protein tagging with ligandable domains.Frontiers in pharmacology · 2025Review
- T cell receptor-directed antibody-drug conjugates for the treatment of T cell-derived cancers.Molecular therapy. Oncology · 2024Article
- Lipoic acid attachment to proteins: stimulating new developments.Microbiology and molecular biology reviews : MMBR · 2024Review
- Tag-free protein modification by lipoate ligase A: exploring substrate tolerance.Analytical sciences : the international journal of the Japan Society for Analytical Chemistry · 2024Article
- Analytical studies on the conjugation site specificity of trastuzumab modified by Escherichia coli lipoate ligase A: multiple-enzyme digestion approach for peptide mapping.Analytical and bioanalytical chemistry · 2023Article
- Conditional activation of an anti-IgM antibody-drug conjugate for precise B cell lymphoma targeting.Frontiers in immunology · 2023Article
- Site-Specific Protein Labeling and Generation of Defined Ubiquitin-Protein Conjugates Using an Asparaginyl Endopeptidase.Journal of the American Chemical Society · 2022Article
- Further assessments of ligase LplA-mediated modifications of proteins in vitro and in cellulo.Molecular biology reports · 2022Article
- Understanding and Engineering Glycine Cleavage System and Related Metabolic Pathways for C1-Based Biosynthesis.Advances in biochemical engineering/biotechnology · 2022Review
- Enzymatic Methods for the Site-Specific Radiolabeling of Targeting Proteins.Molecules (Basel, Switzerland) · 2021Review
- Cell-surface engineering of yeasts for whole-cell biocatalysts.Bioprocess and biosystems engineering · 2021Review
- Strategies for Site-Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags.Chembiochem : a European journal of chemical biology · 2021Review
- Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins.Nature chemistry · 2020Article
- Molecular evolution of peptides by yeast surface display technology.MedChemComm · 2019Review
- From Synthesis to Characterization of Site-Selective PEGylated Proteins.Frontiers in pharmacology · 2019Review
- Recent progress in enzymatic protein labelling techniques and their applications.Chemical Society reviews · 2018Review
- Nanobodies: Chemical Functionalization Strategies and Intracellular Applications.Angewandte Chemie (International ed. in English) · 2018Review
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
Escherichia coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine side chains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides ( Nat. Biotechnol. 2007 , 25 , 1483 - 1487 ) and photo-cross-linkers ( Angew. Chem., Int. Ed. 2008 , 47 , 7018 - 7021 ) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA's utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA's natural protein substrates have a conserved beta-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called "LplA Acceptor Peptide" (LAP). A approximately 10(7) library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a "LAP2" sequence with a k(cat)/K(m) of 0.99 muM(-1) min(-1), >70-fold better than our previous rationally designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483-1487), and only 8-fold worse than the k(cat)/K(m) values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.