Magnetosome Expression of Functional Camelid Antibody Fragments (Nanobodies) in Magnetospirillum gryphiswaldense

Pollithy, Anna; Romer, Tina; Lang, Claus; Müller, Frank-Dietrich; Helma, Jonas; Leonhardt, Heinrich; Rothbauer, Ulrich; Schüler, Dirk

doi:10.1128/aem.05282-11

Cited by 65 publications

(48 citation statements)

References 44 publications

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“…Here, coproduction with DsbC and the sulfhydryl oxidase Erv1P in a tor grx mutant background significantly enhanced the activity of the V HH domain-displaying beads, but it was not absolutely required for production of functional V HH domains. This is in contrast to the above-mentioned study, in which the fusion to DsbC required functional expression of the V HH domain (26,31,32).…”

Section: Discussioncontrasting

confidence: 50%

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Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

Hay¹,

Burr

et al. 2015

Appl Environ Microbiol

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cProof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and V HH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.A ffinity chromatography in various forms has been a cornerstone of protein purification for decades. In its simplest form, the inherent interaction between a target enzyme and its substrate is used, for example, purifying amylase by adsorbing it on insoluble starch. However, this technique is limited by the availability of a naturally occurring interaction and the ability of the substrate to be immobilized on an insoluble matrix (1). A more common and versatile technique is to engineer a tag into the target protein and utilize a protein or matrix with affinity toward that tag (2). Though widely used, this approach is often unacceptable for structural studies and in many commercial production systems due to the chance that the affinity tag may affect the structure, function, or other characteristics of the target protein. Furthermore, the presence of tags is commonly not tolerated in the pharmaceutical industry (3). One approach to combat this is to remove the tag after purification by engineering specific protease cleavage sites or self-cleaving sites such as inteins between the target and the tag, but this introduces further processing and purification steps (to remove the protease and/or cleaved tag) and can often result in a "scar" (residual am...

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Section: Discussioncontrasting

confidence: 50%

“…Recently, a similar approach to produce immobilized binding domains in vivo was used with a red fluorescent protein (RFP)-specific V HH domain on the surface of magnetosomes in Magnetospirillum gryphiswaldense, but no data were supplied regarding the yield or binding capacity (31).…”

Section: Discussionmentioning

confidence: 99%

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

Hay¹,

Burr

et al. 2015

Appl Environ Microbiol

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“…Mutagenesis and recombination of CDR regions to improve the affinity and specificity of sdAb fragments have also been discussed Fanning and Horn 2011). Current studies are focused on the development of new cloning and expression strategies to produce humanised fragments, multivalent constructs and fusions of antibody fragments to other proteins (Saerens et al 2005;Vincke et al 2009;Bell et al 2010;Pollithy et al 2011). Selected articles are presented in Table 4.…”

Section: Production Of Sdab Fragments Using Recombinant Technologymentioning

confidence: 99%

Single-domain antibody fragments derived from heavy-chain antibodies: a review

Eyer¹,

Hruška²

2012

Vet. Med.

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Single-domain antibody (sdAb) fragments derived from heavy-chain antibodies of camelids and cartilaginous fish represent a new generation of therapeutic agents and immunoreagents. Due to their unique characteristics, such as low molecular weight, high physical-chemical stability, good water solubility, and the ability to bind antigens inaccessible to conventional antibodies, they could potentially act as a substitute for conventional therapeutic drugs in the treatment of serious human diseases, and, moreover, could be broadly used in analyses and diagnostics. In this review article, an analysis of 826 publications oriented to heavy-chain antibodies and their sdAb fragments indexed in the Web of Science ® database since 1993 has been carried out. Attention has predominantly been paid to papers published from 2010 to June 2012. Key publications are presented in tables and are characterised by descriptive words, abstracts and references. The presented publications have been sorted according to seven basic criteria: review articles and monographs, heavy-chain antibodies of camelids and sharks, production of sdAb fragments using recombinant technology, characteristic properties of sdAb fragments, application of sdAb fragments in therapy, application of sdAb fragments in diagnostic and immunoanalytical methods and other prospective uses of sdAb fragments. This review article should highlight the typical properties of heavy-chain antibodies and sdAb fragments which differentiate them from conventional antibodies and other available recombinant fragments, and also emphasize their extremely broad application potential, mainly in human disease therapy. At the same time it allows an easy and rapid orientation in numerous publications written on this subject, and facilitates the search for the required data.

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“…2,41 Furthermore, by the combination of nanobody and magnetosome, a VHH-coated magnetosome approach was proposed for in vitro and in vivo immunoprecipitation by magnetical recruitment of antigen partners from cell extracts or within living bacteria. 42 As membranous organelles present in magnetotactic bacteria, magnetosomes contain magnetite particles enabling orientation of bacteria in a magnetic field. 2 By expressing red fluorescent protein (RFP)-binding nanobody with a magnetosome membrane protein MamC, VHHcoated magnetosomes were generated to efficiently recognize and bind their antigens in vitro by magnetically separating …”

Section: Nanobodies As Versatile Research Tools In Biotechnologymentioning

confidence: 99%

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

Wang

Fan

Shao

et al. 2016

IJN

126

107

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Owing to peculiar properties of nanobody, including nanoscale size, robust structure, stable and soluble behaviors in aqueous solution, reversible refolding, high affinity and specificity for only one cognate target, superior cryptic cleft accessibility, and deep tissue penetration, as well as a sustainable source, it has been an ideal research tool for the development of sophisticated nanobiotechnologies. Currently, the nanobody has been evolved into versatile research and application tool kits for diverse biomedical and biotechnology applications. Various nanobody-derived formats, including the nanobody itself, the radionuclide or fluorescent-labeled nanobodies, nanobody homo- or heteromultimers, nanobody-coated nanoparticles, and nanobody-displayed bacteriophages, have been successfully demonstrated as powerful nanobiotechnological tool kits for basic biomedical research, targeting drug delivery and therapy, disease diagnosis, bioimaging, and agricultural and plant protection. These applications indicate a special advantage of these nanobody-derived technologies, already surpassing the “me-too” products of other equivalent binders, such as the full-length antibodies, single-chain variable fragments, antigen-binding fragments, targeting peptides, and DNA-based aptamers. In this review, we summarize the current state of the art in nanobody research, focusing on the nanobody structural features, nanobody production approach, nanobody-derived nanobiotechnology tool kits, and the potentially diverse applications in biomedicine and biotechnology. The future trends, challenges, and limitations of the nanobody-derived nanobiotechnology tool kits are also discussed.

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Magnetosome Expression of Functional Camelid Antibody Fragments (Nanobodies) in Magnetospirillum gryphiswaldense

Cited by 65 publications

References 44 publications

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

Single-domain antibody fragments derived from heavy-chain antibodies: a review

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

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