User:AffiKN/Sandbox

Affibody® molecules
The idea behind developing the Affibody® molecules was to apply a combinatorial protein engineering approach on a small and robust protein scaffold. The aim was to generate new binders capable of specific binding to different target proteins, while retaining the favorable folding and stability properties, and ease of bacterial expression of the parent molecule.

The original Affibody® protein scaffold was designed based on one of the domains of protein A. In contrast to antibodies, Affibody® molecules are composed of alpha helices and lack disulfide bridges. The parent three-helix bundle structure is currently the fastest folding protein structure known.

Affibody® molecules with unique binding properties are acquired by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain. Lately, amino acids outside of the binding surface have been substituted in the scaffold to create a surface entirely different from the ancestral protein A domain.

Specific Affibody® molecules binding a desired target protein can be “fished out” from pools (libraries) containing billions of different variants of the Affibody® protein.

The Affibody® molecules mimic monoclonal antibodies in many ways, and in addition offer several unique properties making them a superior choice for a number of applications.

Key features of Affibody® molecules include:
 * Small size; The size of the Affibody® molecules is only 6 kDa.
 * Stability; Affibody® molecules have been shown to withstand extreme conditions, such as high temperatures (90°C) or acidic and alkaline conditions (at pH 2.5 or pH 11, respectively).
 * Rapid and reversible folding; Affibody® molecules do not contain cysteines or disulfide bridges and fold spontaneously into the correct three dimensional structures. In some studies, temperatures above the melting temperature have been used, with retained binding properties following return to ambient conditions.
 * Chemical synthesis; Synthesized Affibody® molecules fold spontaneously following removal of the protection groups . To date a number of different Affibody® molecules have been successfully produced by chemical synthesis. Also cross-linked variants have been produced.
 * Bacterial production; Affibody® molecules are based on a three-helix bundle domain, which can be easily expressed in soluble and proteolytically stable forms in various host cells on its own or in fusion with other protein partners.
 * Flexible Engineering; Affibody® molecules are very tolerable to modification and are independently folding when incorporated into fusion proteins. Head-to-tail fusions of Affibody® molecules of the same specificity have proven to give avidity effects in target binding, and head-to-tail fusion of Affibody® molecules of different specificities makes it possible to get bi- or multi-specific affinity proteins. Fusions with other proteins are also readily created . Moreover, a site for site-specific conjugation is facilitated by introduction of a unique cysteine at a desired position.
 * High affinity; Binders with an affinity of down to sub-nanomolar have been obtained from naïve library selections, and binders with picomolar affinity have been obtained following affinity maturation.
 * A variety of applications; Affibody® molecules are suitable for a broad range of applications, including protein purification, enzyme inhibition , research reagents for protein capture and detection , diagnostic imaging and targeted therapy.