In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na+-citrate symporter CitS as an example

Huber, Thomas; Steiner, Daniel; Röthlisberger, Daniela; Plückthun, Andreas

doi:10.1016/j.jsb.2007.01.013

Cited by 57 publications

(51 citation statements)

References 32 publications

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“…MsbA was expressed in E. coli C43 (DE3) cells from a pBAD vector and purified using ␤-UDM as detergent. DARPin expression and purification has been described previously (29). Enzymatic biotinylation was facilitated by fusing an Avi-tag sequence to the of MsbA.…”

Section: Methodsmentioning

confidence: 99%

Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Mittal

Böhm

Grütter

et al. 2012

Journal of Biological Chemistry

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Background: ABC exporters are suggested to hydrolyze ATP sequentially implying the existence of asymmetries. Results: An in vitro selected binder (DARPin) was identified that binds to homodimeric MsbA at a stoichiometric ratio of 1:1. Conclusion:The DARPin recognizes asymmetries in MsbA. Significance: Selected binding proteins are useful tools to recognize membrane transporters in novel conformational states.

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Section: Methodsmentioning

confidence: 99%

Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Mittal

Böhm

Grütter

et al. 2012

Journal of Biological Chemistry

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“…Derived from naturally occurring ankyrin repeat proteins, they have been engineered by a consensus design approach. DARPins have been selected from libraries via ribosome or phage display to bind to a wide range of different protein targets (18,(21)(22)(23)(24)(25)(26). DARPins are very well expressed in the cytoplasm of Escherichia coli, monomeric in solution, highly soluble, and very stable; moreover, they can easily be fused to other protein domains to generate multispecific or multivalent DARPins or be chemically conjugated to other molecules (27)(28)(29).…”

Section: The Egf Receptor (Egfr) Has Been Implicated In the Developmentioning

confidence: 99%

“…These DARPins all showed monomeric behavior in size exclusion chromatography; additionally, their affinities were determined to be (sub)nanomolar (23). Using the A431 cell line, which has been developed as a widely used model system for testing anti-EGFR activity (30), we investigated the biological activities of these DARPins and compared them with that of cetuximab.…”

Section: The Egf Receptor (Egfr) Has Been Implicated In the Developmentioning

confidence: 99%

Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling

Boersma

Chao

Steiner

et al. 2011

Journal of Biological Chemistry

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Background:The EGF receptor (EGFR) is an important therapeutic target. Results: Bispecific anti-EGFR designed ankyrin repeat proteins (DARPins), alternative targeting molecules efficiently produced in bacteria, were shown to inhibit A431 cell proliferation and receptor recycling. Conclusion: One bispecific construct containing four DARPins showed a biological activity superior to that of the registered antibody cetuximab. Significance: Bispecific DARPins may form building blocks for tomorrow's cancer therapeutics.

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“…Ligate the PCR fragment into pDST67 (20,25) as fusion with the sequence coding for a N-terminal MRGS(H) 6 tag for purification.…”

Section: Reverse Transcription Of Darpin Mrnamentioning

confidence: 99%

“…They also fold well in the in vitro translation inherent in ribosome display. Using ribosome display DARPins have been evolved to bind various targets with affinities all the way down to the picomolar range (15)(16)(17)(18)(19)(20). In general, probably because of their robust in vitro folding, DARPins are enriched over fewer rounds than antibody fragments.…”

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confidence: 99%

Ribosome Display: A Technology for Selecting and Evolving Proteins from Large Libraries

Dreier

Plückthun

2010

Methods in Molecular Biology

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The selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation. Ribosome display has been used successfully for the selection of antibody fragments and other binding proteins, such as Designed Ankyrin Repeat Proteins (DARPins). Birgit Dreier and Andreas Plückthun AbstractThe selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCRbased methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation. Ribosome display has been used successfully for the selection of antibody fragments and other binding proteins, such as Designed Ankyrin Repeat Proteins (DARPins).

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In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na+-citrate symporter CitS as an example

Cited by 57 publications

References 32 publications

Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling

Ribosome Display: A Technology for Selecting and Evolving Proteins from Large Libraries

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