Биотинмен молекулаларды модификациялау процесі

Biotechnological modification process to attach Biotin to a molecule

In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin (MW = 244.31 g/mol). Biotin binds to streptavidin and avidin with an extremely high affinity, fast on rate, and high specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated to a protein of interest, which allows binding of multiple streptavidin, avidin or neutravidin protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods. Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein protein interactions and post translational events such as ubiquitylation in molecular biology.

Proteins can be biotinylated chemically or enzymatically. Chemical biotinylation utilises various conjugation chemistries to yield nonspecific biotinylation of amines, carboxylates, sulfhydryls and carbohydrates (e. g., NHS coupling gives biotinylation of any primary amines in the protein). Enzymatic biotinylation results in biotinylation of a specific lysine within a certain sequence by a bacterial biotin ligase. Most chemical biotinylation reagents consist of a reactive group attached via a linker to the valeric acid side chain of biotin. As the biotin binding pocket in avidin / streptavidin is buried beneath the protein surface, biotinylation reagents possessing a longer linker are desirable, as they enable the biotin molecule, once it has been attached to its target, to be more accessible to binding avidin/streptavidin/Neutravidin protein. This linker can also mediate the solubility of biotinylation reagents; linkers that incorporate poly(ethylene) glycol (PEG) can make water insoluble reagents soluble or increase the solubility of biotinylation reagents that are already soluble to some extent.

In contrast to chemical biotinylation methods, enzymatic biotinylation allows biotin to be linked at exactly one residue present in the protein. This biotinylation reaction can also go to completion, meaning that the product is generated with high uniformity and can be linked to streptavidin in a defined orientation e. g. for MHC multimers. Enzymatic biotinylation is most often carried out by E. coli biotin holoenzyme synthetase, also known as biotin ligase (BirA, ). The most common way of targeting a protein of interest is by fusing the protein at its N terminus, C terminus or at an internal loop to a 15 amino acid peptide , termed AviTag or Acceptor Peptide (AP). Once tagged, the protein is then incubated with BirA allowing biotinylation to take place in the presence of biotin and ATP. Enzymatic biotinylation can also take place in vivo typically through the co expression of an Avitag tagged protein and BirA. The natural substrate of BirA is the biotin carboxyl carrier protein (BCCP). Before smaller tags were discovered, a protein needed to be fused to the entire BCCP to be targeted. A protein fused by BCCP can be recognized by biotin molecules in vivo and attach to it. A few other small tags have been used before AviTag, but AviTag is the most efficient so far. Upon the standard deprotection, the conjugates obtained can be purified using reverse phase or anion exchange HPLC

Photoactivatable biotinylation reagents are ideal when primary amines, sulfhydryls, carboxyls and carbohydrates are not available for labeling. These reagents rely on aryl azides, which become activated by ultraviolet light (UV; >350 nm), which then react at C H and N H bonds. Because these types of bonds occur independent of the type of amino acid, this type of biotinylation is termed "non specific". Photoactivatable biotinylation reagents can also be used to activate biotinylation at specific times in an experiment or during certain reaction conditions, by simply exposing the reaction to UV light at the specific time or condition.

The biotin tag can be used in affinity chromatography together with a column that has avidin (or streptavidin or neutravidin) bound to it, which is the natural ligand for biotin. However, harsh conditions (e. g., 6M GuHCl at pH 1.5) are needed to break the avidin/streptavidin biotin interaction, which will most likely denature the protein carrying the biotin tag. If isolation of the tagged protein is needed, it is better to tag the protein with iminobiotin. This biotin analogue gives strong binding to avidin/streptavidin at alkaline pH, but the affinity is reduced upon lowering the pH. Therefore, an iminobiotin tagged functional protein can be released from an avidin/streptavidin column by decreasing the pH (to around pH 4).

This tag can also be used in detection of the protein via anti biotin antibodies or avidin/streptavidin tagged detection strategies such as enzyme reporters (e. g., horseradish peroxidase, alkaline phosphatase) or fluorescent probes. This can be useful in localization by fluorescent or electron microscopy, ELISA assays, ELISPOT assays, western blots and other immunoanalytical methods. Detection with monovalent streptavidin can avoid clustering or aggregation of the biotinylated target.

The non covalent bond formed between biotin and avidin or streptavidin has a binding affinity that is higher than most antigen and antibody bonds and approaches the strength of a covalent bond. This very tight binding makes labeling proteins with biotin a useful tool for applications such as affinity chromatography using immobilized avidin or streptavidin to separate the biotinylated protein from a mixture of other proteins and biochemicals. Biotinylated protein such as biotinylated bovine serum albumin (BSA) is used in solid phase assays as a coating on the well surface in multiwell assay plates. Biotinylation of red blood cells has been used as a means of determining total blood volume without the use of radiolabels such as chromium 51, allowing volume determinations in low birth weight infants and pregnant women who could not otherwise be exposed to the required doses of radioactivity. Furthermore, biotinylation of MHC molecules to create MHC multimers has become a useful tool for identifying and isolating antigen specific T cell populations. More recently, in vivo protein biotinylation was developed to study protein protein interactions and proximity in living cells

Reaction conditions for biotinylation are chosen so that the target molecule (e. g., an antibody) is labeled with sufficient biotin molecules to purify or detect the molecule, but not so much that the biotin interferes with the function of the molecule.

The HABA (2 (4 hydroxyazobenzene) benzoic acid) assay can be used to determine the extent of biotinylation. HABA dye is bound to avidin or streptavidin and yields a characteristic absorbance. When biotinylated proteins or other molecules are introduced, the biotin displaces the dye, resulting in a change in absorbance at 500 nm. This change is directly proportional to the level of biotin in the sample. The disadvantage of the HABA assay is that it uses large amounts of sample.

Extent of biotinylation can also be measured by streptavidin gel shift, since streptavidin remains bound to biotin during agarose gel electrophoresis or polyacrylamide gel electrophoresis. The proportion of target biotinylated can be measured via the change in band intensity of the target with or without excess streptavidin, seen quickly and quantitatively for biotinylated proteins by Coomassie brilliant blue staining.