Affitins (commercial name Nanofitins) are artificial

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s with the ability to selectively bind

antigen In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...

s. They are structurally derived from the

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

binding protein Sac7d, found in ''

Sulfolobus ''Sulfolobus'' is a genus of microorganism in the family Sulfolobaceae. It belongs to the kingdom Thermoproteati of the Archaea domain. ''Sulfolobus'' species grow in volcanic springs with optimal growth occurring at pH 2–3 and temperatu ...

acidocaldarius'', a microorganism belonging to the

archaeal Archaea ( ) is a domain of organisms. Traditionally, Archaea only included its prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even though the domain Archaea cladis ...

domain. By randomizing the

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various targets, such as

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

s, proteins, viruses, and bacteria. Affitins are

antibody mimetic Antibody mimetics are organic compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. (Antibo ...

s and are being developed as an alternative to

antibodies An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that caus ...

as tools in

biotechnology Biotechnology is a multidisciplinary field that involves the integration of natural sciences and Engineering Science, engineering sciences in order to achieve the application of organisms and parts thereof for products and services. Specialists ...

. They have also been used as specific inhibitors for various enzymes. Affitins can be utilized in biochemical purification techniques, specifically in affinity chromatography. The ability of Affitins to selectively bind antigens is used to target specific proteins. Scientists have been able to purify human

immunoglobulin G Immunoglobulin G (IgG) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG ...

(hIgG), bacterial PulD protein, and chicken egg lysozyme using Affitin columns with a high degree of purity. These have the ability to act as specific ligands for the proteins of interest that are needed when the fusion of proteins to polypeptide tags is impossible or carries no advantage, and thus build affinity columns as is the case in the production of biopharmaceuticals. They were immobilized on an

agarose Agarose is a heteropolysaccharide, generally extracted from certain red algae. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is on ...

matrix and the columns had a high degree of selectivity. In addition to this, antibodies and non-immunoglobin proteins can be purified by using affitins via affinity chromatography. Due to their small size and high solubility, they can be easily produced in large amounts using bacterial expression systems.

Properties

Affitins consist of 66 amino acids and have a

molecular mass The molecular mass () is the mass of a given molecule, often expressed in units of daltons (Da). Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The derived quan ...

of about 7

kDa The dalton or unified atomic mass unit (symbols: Da or u, respectively) is a unit of mass defined as of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state and at rest. It is a non-SI unit accepted f ...

; this is small compared to antibodies with some 130–150 kDa. Obtained from a

thermophile A thermophile is a type of extremophile that thrives at relatively high temperatures, between . Many thermophiles are archaea, though some of them are bacteria and fungi. Thermophilic eubacteria are suggested to have been among the earliest bacte ...

organism, they are unusually heat resistant proteins. In addition, Affitins are durable – they are able to withstand many cycles of purification. Unlike antibodies, affitins are produced ''in vitro'', and therefore can be generated more quickly. Due to their small size and high solubility, they can easily be produced in large amounts using bacterial expression systems. Affitins are strongly modified reagents that are extremophilic since they are found in Archae like Sac7d, which is a hyperthermostable protein. They are artificially binding proteins with high affinity, small size, and low structural complexity. They have two different modes of binding. The first requires a flat surface whereas the second mode of binding requires a flat surface and two short loops. They are thermally and chemically stable reagents and their stability can be further increased by using mutation or grafting techniques. Other methods of stabilizing them include the use of sequence elements from other proteins that belong to the same family, switching a binding surface, and thus, have longer binding capacities. This was done by grafting the binding surface of D1Sac7d onto Sso7d, which is more stable, and by introducing point mutations previously identified as stabilizing for WT Sso7d.

References

{{Engineered antibodies Antibody mimetics