title: SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides for in vitro applications.
abstract: Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Ulrich
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.|EMBL Australia, Monash University, Clayton, Victoria, 3800, Australia.|The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, 3800, Australia.
authors.forename: Max J
authors.identifier: http://orcid.org/0000-0002-9739-6157
authors.initials: MJ
authors.lastname: Cryle
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry; D060326:Chemistry Techniques, Synthetic; D002733:Chlorophenols / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006020:Glycopeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D002733:Chlorophenols; D003432:Cross-Linking Reagents; D006020:Glycopeptides; D010455:Peptides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; C023490:complestatin; D019881:Aminoacyltransferases; D010453:Peptide Synthases
keywords: biosynthetic enzymes; fusion; ligation; peptide; protein