Biotinylation: Application & Overview

Biotinylation: Overview

Fusion tags are widely used in labs for the identification and isolation of recombinant proteins that are expressed in bacteria or other cells (such as E.coli, plants, or mammalian cells). Out of many, one of the most commonly used ones is the Biotin tag. 

Biotin is a small protein of molecular weight 244.31. It’s used for the synthesis of valine, isoleucine, and fatty acids and in the gluconeogenesis process. It specifically binds to streptavidin and avidin at a fast rate and forms a complex with an extremely high affinity (Kd, ~ 10-14 mol/L). The process of biotin labeling is known as biotinylation.

Figure: Biotin structure

Protein biotinylation involves covalently attaching biotin molecules to other biomolecules, such as antibodies, peptides, proteins, nucleic acids (DNA and RNA), oligonucleotides, and others. It’s a fast and specific reaction that does not alter the natural roles of biomolecules.

Figure: Binding of biotinylated antibodies with avidin or streptavidin.

The biotin complexes formed with some biomolecules, such as avidin (which has multiple biotin-binding sites), are even stable under extreme physiological conditions like high temperature, high/low pH, and high salt concentrations.

One advantage of biotinylation is that the biotin molecules have the affinity to bind to more than one protein molecule. As a result, the protein of interest can be detected with greater sensitivity. Today, many types of biotinylation reagents are available for use in different labeling techniques. 

Biotinylated protein purification is extensively exploited in molecular biology and biotech labs to study post-translational modification, such as ubiquitylation, and protein-protein interaction.

In this article, we will cover the working mechanism of Biotin, its uses in protein and affinity purification, and applications in different biotech and pharma industrial sectors. 

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How Does Biotinylation Work?

Biotinylation can be performed either through chemical or enzymatic means. Chemical methods (such as sulfo-NHS- coupling) can be performed in vitro or in vivo and provide greater flexibility than the enzymatic method.

Figure: Reaction between Biotin-NHS and Lysine.

The enzymatic methods involve the co-expression of an exogenously expressed protein and bacterial biotin ligase (in E.coli known as BirA). This method offers more uniform biotinylation (at the expense of ATP) than the chemical method.

One example of an enzymatic approach is the use of 13 amino acid sequences (termed as AviTag). It helps in endogenous BirA recognition and biotin labeling to the N-terminal of the protein. The process involves cloning the gene of the protein of interest with a plasmid containing a gene for BirA expression. After biotinylation, proteins can be identified by mass spectrometry.

(Confirm the recombinants using PCR and agarose gel electrophoresis technique and allow biotin to come at room temperature before using them in the experiments.) 

Biotinylating Groups

By biotinylating a protein of interest, one aims to label it without significantly altering the protein’s normal biological function. However, if the biotinylation reagent attaches to amino acids regulating protein activity (such as substrate binding), it can interfere with protein functions. 

This interference can be reduced by targeting different specific amino acid functional groups, such as:

  • Primary amines (-NH2) — Located in the side chain of lysine (Lys, K) residues and at the N-terminus of each polypeptide chain. 
  • Carboxyls (-COOH) — Present at the C-terminus of each polypeptide chain and in the side chains of glutamic acid (Glu, E) and aspartic acid (Asp, D). It needs a buffer without amine (such as tris and glycine) and carboxyls, such as citrate and acetate.
  • Sulfhydryls (-SH) — Located in the side chain of cysteine (Cys, C). 
  • Carbonyls (-CHO) — Created by oxidizing carbohydrate groups in glycoproteins.
  • Non-selective groups: The photoactivatable biotinylation reagents are used to label proteins without function groups or at specific times in the incubation process.

Fusion tags can be detached from proteins of interest by cleaving a linker region using a protease that does not cleave the target protein.

What is Biotinylation Used For?

Biotinylation or biotin-streptavidin and avidin interaction are extensively exploited in the identification and separation of target cells/antigens. The applications include:

  • Immunoassays (ELISAs and Western Blotting)
  • Affinity chromatography
  • Pull-down assays
  • Supramolecular construction
  • Targeting cancer cells for drug

A number of factors must be carefully considered and optimized before choosing the appropriate biotinylation reagents and protein of interest, such as:

  • Solubility: The solubility of biotinylation reagents can influence the solubility of biotinylated proteins by allowing access to target proteins in hydrophobic or hydrophilic environments.
  • Spacer arm length: The length of the spacer arm affects biotin availability for avidin binding.
  • Cleavability/reversibility: the captured biotinylated proteins can be eluted by cleaving biotin molecules from the target protein or reverse biotin-avidin interactions. It enables the selective removal of biotinylated molecules from biotin-binding proteins.

Protein Purification

Magnetic beads and biotin-(strept)-avidin interaction have become increasingly popular for protein/cell separation applications. 

The high affinity of biotin for avidin/streptavidin enables researchers to purify protein even in harsh conditions, such as extreme pH and temperature or the presence of denaturing agents, without affecting target proteins during elution.

Affinity Protein Purification

Affinity chromatography can be performed using an avidin-bound column for biotin-tagged proteins—avidin is a natural ligand of biotin. However, rough conditions (e.g., 6M GuHCl at pH 1.5) are required to break the avidin-biotin bonds that can often denature target proteins. Thus, in such a case, imino-biotinylation of the proteins can be a savior. 

Iminobiotin is an analog of biotin that strongly binds to avidin at alkaline pH. However, at lower pH (around pH 4), iminobiotin-tagged functional proteins can be easily released without harming target proteins.

What Industries Use Biotinylation?  

Biotinylation is a process routinely used in labs to label proteins with biotin tags. The tag help researchers to detect and purify target proteins using avidin conjugates in variant workflows, such as immunohistochemistry (IHC), western blot analysis, immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and others. Further, it also has applications in cell surface labeling and flow cytometry/fluorescence-activated cell sorting (FACS). 

Pharmaceutical 

Avidin-biotin covalent interaction is gaining popularity in pharma industries, especially in nanoscale drug delivery systems for agents like small molecules, nucleic acids, and proteins. The system offers easy fabrication without disturbing the biological and chemical properties of the coupled groups. Further, the system is also being researched in diagnosing surface antigens and different tumors. 

Immunology 

Biotinylation is exploited heavily in affinity purification and solid-phase immunoassays  For example, biotinylated antibodies are used in a range of immunochemical assays for signal amplification purposes. Further, many immunological systems exploited the biotin-avidin interaction for the isolation, localization, and visualization of various antigens.

Histochemistry

Biotinylation has major applications in receptor localization and FRET-based flow cytometry. One interesting aspect in histochemical studies is that the staining intensity depends on the enzymatic activity — the more enzymes bound to tissues, the greater the staining intensity.

Thus, using tetravalent streptavidin and biotinylated antibodies (bound to the antigen) to achieve this amplification is ideal. The Avidin–Biotin Complex (ABC) and the Labeled Streptavidin–Biotin (LSAB) staining methods ate the two most frequently used techniques that provide the best signal amplification for IHC.

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Biotinylation is a process of attaching biotin tags to molecules like peptides, proteins, carbohydrates, and nucleic acids for studies like protein-protein interaction and protein purification.

The process has major applications in pharmaceutical, biotechnology, and immunology areas in a range of workflows, such as western blotting, immunohistochemistry, and affinity purification.

Assays like this require researchers to use high-quality and pure biotinylated reagents combined with high-throughput equipment. It enables you to obtain accurate and validated results without multiple repetitions of the experiments.

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