Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions
Carla P. Guimaraes,Martin D. Witte,Christopher S. Theile,Gunes Bozkurt,Lenka Kundrat,Annet E M Blom,Hidde L. Ploegh +6 more
TLDR
In this paper, the authors describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences and provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site.Abstract:
Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.read more
Citations
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Crystal structure of a substrate-engaged SecY protein-translocation channel
TL;DR: In this article, a crystal structure of the active SecY channel, assembled from the SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA, is presented.
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Intra-Spike Crosslinking Overcomes Antibody Evasion by HIV-1
Rachel P. Galimidi,Joshua S. Klein,Maria S. Politzer,Shiyu Bai,Michael S. Seaman,Michel C. Nussenzweig,Anthony P. West,Pamela J. Bjorkman,Pamela J. Bjorkman +8 more
TL;DR: The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-SpikeCrosslinking for therapy or passive protection.
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Noninvasive imaging of tumor progression, metastasis, and fibrosis using a nanobody targeting the extracellular matrix.
Noor Jailkhani,Jessica R. Ingram,Mohammad Rashidian,Steffen Rickelt,Chenxi Tian,Howard Mak,Zhigang Jiang,Hidde L. Ploegh,Richard O. Hynes +8 more
TL;DR: NJB2, a nanobody specific for an alternatively spliced domain of fibronectin expressed in disease ECM and neovasculature, is described and shown by noninvasive in vivo immuno-PET/CT imaging that NJB2 detects primary tumors and metastatic sites with excellent specificity in multiple models of breast cancer, including human and mouse triple-negative breast cancer and in melanoma.
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Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics
TL;DR: The fundamental technical aspects of single-molecule fluorescence are introduced and it is illustrated how the optical techniques and the labeling schemes have been combined to investigate GPCR signaling and dynamics at the single-Molecule level.
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Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity
Koen Wagner,Mark J. Kwakkenbos,Yvonne Claassen,Kelly Maijoor,Martino Bohne,Koenraad F. van der Sluijs,Martin D. Witte,Diana van Zoelen,Lisette A. H. M. Cornelissen,Tim Beaumont,Arjen Q. Bakker,Hidde L. Ploegh,Hergen Spits +12 more
TL;DR: A format for bispecific antibodies, fusing two full-sized antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer is presented.
References
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Arnaud Gautier,Alexandre Juillerat,Christian Heinis,Ivan R. Corrêa,Maik Kindermann,Florent Beaufils,Kai Johnsson +6 more
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