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Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.
TLDR
Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. Expand
Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.
TLDR
The efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins, which have the properties required for biotechnological applications, is described. Expand
Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.
TLDR
The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes. Expand
Assembly of a functional immunoglobulin Fv fragment in Escherichia coli.
TLDR
An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli, and experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the FV fragment, and that there are no inactive protein in the preparation. Expand
Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins.
TLDR
The folding thermodynamics and kinetics of ankyrin repeat proteins were analyzed both by classical two- state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. Expand
Stability engineering of antibody single-chain Fv fragments.
TLDR
Progress in rational and evolutionary engineering methods, the structural implications of these results, as well as some examples where stability engineering has been successfully applied are summarized. Expand
Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size.
TLDR
It is shown that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), limits can be overcome on delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life,such as PEGylated Darpins, in which the importance of affinity is less pronounced. Expand
Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.
TLDR
The structure of the fusion protein presented here clearly shows that, during the infection process, the g3p N2 domain is displaced by the TolA D3 domain, which is not similar to any previously known protein motifs. Expand
Structure of a kinesin–tubulin complex and implications for kinesin motility
TLDR
The X-ray structure of human kinesin-1 bound to αβ-tubulin is determined, which defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the kinesIn motor domain from microtubules. Expand
High-affinity binders selected from designed ankyrin repeat protein libraries
TLDR
The evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding and combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces are generated, an attractive alternative to antibody libraries. Expand
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