Affilins are genetically engineered protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s with the ability to selectively bind antigen

Antigen

An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...

s. They are structurally derived from one of two proteins, gamma-B crystallin or ubiquitin

Ubiquitin

Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...

, both occurring in humans. Affilins are constructed by modification of near-surface amino acid

Amino acid

Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

s of these proteins and isolated by display techniques such as phage display

Phage display

Phage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P...

. They resemble antibodies in their affinity to antigens but not in structure, which makes them a type of antibody mimetic

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...

. Affilins are being developed as potential new biopharmaceutical

Biopharmaceutical

Biopharmaceuticals are medical drugs produced using biotechnology. They include proteins , nucleic acids and living microorganisms like virus and bacteria where the virulence of viruses and bacteria is reduced by the process of attenuation, they can be used for therapeutic or in vivo diagnostic...

 drugs.

Structure

Two proteins, gamma-B crystallin and ubiquitin, have been described as scaffolds for affilins. Certain amino acids in these proteins can be substituted by others without losing structural integrity, a process creating regions capable of binding different antigens, depending on which amino acids are exchanged. In both types, the binding region is located in a beta sheet

Beta sheet

The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...

 structure, whereas the binding regions of antibodies, called complementarity determining region

Complementarity determining region

Complementarity determining regions are regions within antibodies or T cell receptors where these proteins complement an antigen's shape. Thus, CDRs determine the protein's affinity and specificity for specific antigens...

s, are flexible loops.

Based on gamma crystallin

Gamma crystallin is a family of proteins found in the eye lens of vertebrate

Vertebrate

Vertebrates are animals that are members of the subphylum Vertebrata . Vertebrates are the largest group of chordates, with currently about 58,000 species described. Vertebrates include the jawless fishes, bony fishes, sharks and rays, amphibians, reptiles, mammals, and birds...

s, including humans. It consists of two identical domains

Protein domain

A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...

 with mainly beta sheet structure and a total molecular mass of about 20 kDa

KDA

KDA may refer to:* Karachi Development Authority* Kongsberg Defence & Aerospace* Kotelawala Defence Academy* Kramer Design Associates* Lithium diisopropylamide, KDA is the potassium analogue of lithium diisopropylamideOr kDa may refer to:...

. The eight near-surface amino acids 2, 4, 6, 15, 17, 19, 36, and 38 are suitable for modification.

Based on ubiquitin

Ubiquitin, as the name suggests, is a highly conserved protein occurring ubiquitously in eucaryotes. It consists of 76 amino acids in three and a half alpha helix

Alpha helix

A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...

 windings and five strands constituting a beta sheet. The eight surface-near exchangeable amino acids 2, 4, 6, 62, 63, 64, 65, and 66 are located at the beginning of the first N-terminal beta strand (2, 4, 6), at the nearby beginning of the C-terminal strand and the loop leading up to it (63–66). The resulting affilins are about 10 kDa in mass.

Properties

The molecular mass of crystallin and ubiquitin based affilins is only one eighth or one sixteenth of an IgG antibody, respectively. This leads to an improved tissue permeability, heat stability up to 90 °C (195 °F), and stability towards acids and bases

Base (chemistry)

For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

. The latter enables affilins to pass through the intestine

Intestine

In human anatomy, the intestine is the segment of the alimentary canal extending from the pyloric sphincter of the stomach to the anus and, in humans and other mammals, consists of two segments, the small intestine and the large intestine...

, but like most proteins they are not absorbed into the bloodstream. Renal clearance

Clearance (medicine)

In medicine, the clearance is a measurement of the renal excretion ability. Although clearance may also involve other organs than the kidney, it is almost synonymous with renal clearance or renal plasma clearance. Each substance has a specific clearance that depends on its filtration characteristics...

, another consequence of their small size, is the reason for their short plasma half-life

Biological half-life

The biological half-life or elimination half-life of a substance is the time it takes for a substance to lose half of its pharmacologic, physiologic, or radiologic activity, as per the MeSH definition...

, generally a disadvantage for potential drugs.

Production

A molecular library of affilins is generated by random mutagenesis

Mutagenesis

Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures...

. Substituting some or all of the eight amino acids at the potential binding site with one of the nineteen proteinogenic amino acid

Proteinogenic amino acid

Proteinogenic amino acids are those amino acids that can be found in proteins and require cellular machinery coded for in the genetic code of any organism for their isolated production. There are 22 standard amino acids, but only 21 are found in eukaryotes. Of the 22, 20 are directly encoded by...

s excluding cysteine

Cysteine

Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...

 gives 19⁸ ≈ 17,000,000,000

1000000000 (number)

1,000,000,000 is the natural number following 999,999,999 and preceding 1,000,000,001.In scientific notation, it is written as 109....

 possible combinations. Cystein is excluded because of its liability to form disulfide bond

Disulfide bond

In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

s. In a dimer

Protein dimer

In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...

ic affilin, up to 14 amino acids are exchanged, resulting in 8 × 10¹⁷ combinations, but not all of these are realised in a given library.

The next step is the selection of affilins that bind the desired target protein. To this end display techniques such as phage display or ribosome display

Ribosome display

Ribosome display is a technique used to perform in vitro protein evolution to create proteins that can bind to a desired ligand. The process results in translated proteins that are associated with their mRNA progenitor which is used, as a complex, to bind to an immobilized ligand in a selection step...

 are used. The fitting species are isolated and characterised physically, chemically and pharmacologically. Subsequent dimerisation or multimerisation can increase plasma half-life and, due to avidity

Avidity

In proteins, avidity is a term used to describe the combined strength of multiple bond interactions. Avidity is distinct from affinity, which is a term used to describe the strength of a single bond...

, affinity to the target protein. Radionuclide

Radionuclide

A radionuclide is an atom with an unstable nucleus, which is a nucleus characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or to an atomic electron. The radionuclide, in this process, undergoes radioactive decay, and emits gamma...

s or cytotoxins can be conjugated to affilins, making them potential tumour therapeutics and diagnostics. Conjugation of cytokine

Cytokine

Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...

s has also been tested in vitro

In vitro

In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...

.

Large-scale production of affilins is facilitated by E. coli and other organisms commonly used in biotechnology.

External links

• Scil Proteins, the developer