Broadpharm知識課堂什么是生物素化？What is Biotinylation?

生物素（Biotin）為B族維生素之一，又稱維生素H、維生素B7、輔酶R（Coenzyme R）等。生物素是一種水溶性維生素，屬于B族維生素的一種。

生物素化，也稱為生物素標(biāo)記，是將生物素共價連接到生物分子（如蛋白質(zhì)、抗體、肽、寡核苷酸和其他大分子）的過程。該反應(yīng)是快速、特異性的，并且由于生物素體積小 （MW = 244.31） 而不太可能干擾生物分子的自然功能。

生物素與鏈霉親和素和親和素特異性結(jié)合，形成具有強(qiáng)親和力 （Kd， ~ 10-14 mol/L） 和快速接通速率的復(fù)合物。即使在高/低 pH 值、高溫、高鹽濃度等惡劣條件下，復(fù)合物也非常穩(wěn)定。

生物素-親和素和鏈霉親和素系統(tǒng)廣泛用于靶抗原/細(xì)胞的檢測和分離。這些應(yīng)用包括免疫測定（ELISA 和 Western 是常規(guī)應(yīng)用）、親和色譜、沉降測定、超分子構(gòu)建、靶向癌細(xì)胞進(jìn)行藥物遞送等。最近，基于生物素-（strept）親和素相互作用和磁珠的蛋白質(zhì)/細(xì)胞分離的應(yīng)用需求不斷增加。

Broadpharm知識課堂什么是生物素化？What is Biotinylation?

Biotinylation, also known as biotin labeling, is the process of covalently attaching biotin(s) to biomolecules: such as proteins, antibodies, peptide, oligonucleotide, and other macromolecules. The reaction is rapid, specific and is unlikely to disturb the natural function of the biomolecules due to the small size of biotin (MW = 244.31).

Biotin specifically binds to streptavidin and avidin to form a complex with an extremely high affinity (Kd, ~ 10^-14 mol/L) and fast on-rate. The complexes are very stable under even extreme conditions such as high/low pH, high temperature, high salt concentrations, etc.

Biotin-avidin and streptavidin systems are widely used in detection and separation of target antigens/cells. These applications include immunoassays (ELISAs and Westerns being the most popular applications), affinity chromatography, pull-down assays, supramolecular construction, targeting of cancer cells for drug delivery, and many others. Recently, there are increasing application demands for protein/cell separation based on biotin-(strept)avidin interaction and magnetic beads.

Biotinylation Chemistry

For biotinylation chemistry, the most common reactions involve amines with biotin-NHS ester and click chemistry of azide with an alkyne (e.g. DBCO), as shown in Figure 2.

Biotinylation Reagent in Biotin Labeling

The selection of biotinylation reagent should consider a few factors: target functional group, water solubility, cell membrane permeability, cleavability, and length of the reagent.

The functional groups can be click chemistry reactive, amine reactive, carbonyl reactive, carboxyl reactive, and sulfhydryl reactive (Figure 3). There are also reversible and cleavable biotinylation reagents to help with the specific elution of biotinylated proteins.

Pegylated biotin reagents are particularly attractive due to their water solubility, no toxicity, and low immunogenic properties. Monodispersed PEGs have a well-defined chain length, allowing for specific biotin-based complexes to be designed and studied.

In addition, BroadPharm provides desthiobiotin products for special application, such as affinity purification. Desthiobiotin is a modified form of biotin that binds less tightly to avidin and streptavidin than biotin while still providing excellent specificity. Unlike biomolecules that are labeled with biotin, proteins and other targets labeled with desthiobiotin can be eluted under a soft, mild elute conditions to avoid denaturing the protein of interest.

BroadPharm is a leader of biotinylation reagents that help advance our customer's research, and offers a variety of pegylated and non-pegylated biotinylation reagents to meet your requirement.

Journal Reference:

1. Cull and Schatz, "Biotinylation of proteins in vivo and in vitro using small peptide tags", Methods in Enzymology, 326, (2000): 430-440

2. Minde, et al., "Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions", Communications Biology, 3, 38, (2020): 1-13