title: Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldense.
abstract: Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.
authors.affiliation: Ludwig-Maximilians-Universität München, Dept. Biologie I, Bereich Mikrobiologie, Biozentrum der LMU, Großhaderner Str. 2-4, D-82152 Martinsried, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Pollithy
authors.forename: Tina
authors.initials: T
authors.lastname: Romer
authors.forename: Claus
authors.initials: C
authors.lastname: Lang
authors.forename: Frank D
authors.initials: FD
authors.lastname: Müller
authors.forename: Jonas
authors.initials: J
authors.lastname: Helma
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
authors.forename: Dirk
authors.initials: D
authors.lastname: Schüler
mesh_terms: D000818:Animals; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D058185:Magnetite Nanoparticles; D055944:Magnetosomes / Q000378:metabolism*; D042201:Magnetospirillum / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D007128:Immunoglobulin Fragments; D008164:Luminescent Proteins; D058185:Magnetite Nanoparticles; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein