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Biotinylation With dPEG® Products

Biotinylation is the process of modifying a biomolecule with biotin, a B vitamin also known as vitamin H. Currently, biotinylation of proteins, peptides, and other biomolecules is one of the most popular bioconjugation reactions used. Biotin has an extraordinary affinity for avidin from egg white. In fact, the biotin-avidin affinity (Ka ≈ 1015 M-1) is the strongest non-covalent affinity presently known. In addition, the affinity between wild-type streptavidin from Streptomyces avidinii and biotin (Ka ≈ 1013 M-1) is nearly as strong as the avidin-biotin affinity.

Today, avidin-biotin and streptavidin-biotin systems are used in a huge array of different applications. These applications include immunoassays (ELISAs and Westerns are the most popular applications), affinity chromatography, pull-down assays, supramolecular construction, pretargeting of cancer cells for drug delivery, and many others. The range of applications using biotin-streptavidin technology is quite broad. Importantly, avidin and streptavidin are largely interchangeable in function. Following Hermanson (see reference 1, page 466), we use the term (strept)avidin to denote that either protein can be used.


Avidin and Streptavidin


Researchers discovered avidin in hen egg whites during the 1920's while working to isolate biotin. Avidin is a tetrameric glycoprotein. Each subunit is approximately 16.4 kDa. The complete protein has an approximately 66 kDa molecular weight. Avidin is a highly stable enzyme. It resists denaturation with 8-M urea or 3-M guanidine hydrochloride. When complexed to biotin, the avidin-biotin complex is even more stable.

With an isoelectric point (pI) of 10 and high carbohydrate content, avidin binds non-specifically with cellular components other than biotin. For example, the protein's strong positive charge can cause ionic interactions with negatively charged molecules on cell surfaces. This is a disadvantage with highly sensitive assays. Consequently, streptavidin has generally replaced avidin in most assays.



Discovered in 1963 in culture broths of the bacterium Streptomyces avidinii, streptavidin is a non-glycosylated tetrameric protein. Its pI is much lower (pI = 5 – 6) than avidin. Moreover, its primary structure has only about 30% sequence homology with avidin. Nevertheless, the secondary, tertiary, and quaternary structures of streptavidin are nearly identical to the equivalent structures of avidin. Consequently, the binding affinity of streptavidin for biotin is almost as strong as the affinity of avidin for biotin. See, Figure 1, showing the structure of tetrameric streptavidin with bound biotin.

Cartoon ribbon structure of the biotin-binding protein streptavidin, which is produced by the bacterium Streptomyces avidinii, containing two molecules of biotin (represented by space-filling spheres) bound to the protein.
Figure 1: Tetrameric Streptavidin ribbon structure with two molecules of biotin (atoms represented by space-filling spheres) bound to the protein. Credit: Oxford grad at English Wikipedia [CC BY-SA 3.0 (], via Wikimedia Commons.
Streptomyces avidinii processes streptavidin after synthesis, trimming the N- and C-terminal portions of each monomer to a core monomer of about 13.2 kDa. Therefore, a post-processed streptavidin tetramer has a molecular weight of about 52.8 kDa.

Biotinylation with dPEG® Products is Superior to Biotinylation with Traditional Products

Traditional biotinylation involves either the direct coupling of biotin to a target molecule or the coupling of biotin to a spacer molecule such as lysine (forming biocytin), aminocaproic acid, an aliphatic diamine (e.g., butylene diamine, hexylene diamine), or some similar linker, followed by coupling of the biotin-spacer conjugate to the biomolecule of interest. The spacer provides better access to the relatively deep biotin binding pockets of avidin and streptavidin.

Although ostensibly a water soluble vitamin, biotin is actually poorly soluble in water. Spacers that are traditionally coupled to biotin are quite hydrophobic. Adding these hydrophobic spacers to poorly-water-soluble biotin creates a water insoluble molecule. See Figure 2.



Disadvantages of Traditional Hydrophobic Biotinylation Reagents

The use of hydrophobic spacers with biotin has several disadvantages. Firstly, the number of biotin molecules that can be conjugated onto a protein is limited. Excessive conjugation of biotin containing a hydrophobic linker or spacer to a protein results in aggregation and precipitation of the biotinylated protein. Savage et al., recommend targeting 2-3 biotins per polyclonal IgG with NHS esters of LC-biotin (page 86-87). Secondly, even with low substitution levels of LC-Biotin, a biotinylated protein is less stable in solution. Over time, the protein may both lose activity and aggregate (Hermanson, ref. 1, page 487). Thirdly, because traditional biotinylation reagents are hydrophobic, the reagents cannot be dissolved conveniently in aqueous media. Rather, the biotinylation reagents must be dissolved in a water-miscible organic solvent such as DMAC, DMF, or DMSO and then added to the reaction.


Advantages of Discrete PEG (dPEG®) Biotinylation Reagents

In contrast to traditional biotinylation products, biotin modified by the addition of just a short, discrete PEG — both dPEG®4 and dPEG®12 have been tested in-house — demonstrates remarkably good water solubility. This is due in part to the fact that amphiphilic PEG is able to coordinate two to three molecules of water per ethylene oxide unit along the PEG chain (see, Fee 2010; Antonsen, 1992).

In addition to increasing the water solubility of molecules to which it is conjugated, dPEG® products reduce non-specific binding and reduce or eliminate aggregation of biomolecules. Quanta BioDesign demonstrated elimination of aggregation in a 2011 poster presentation. The reduction of non-specific binding by PEG-modified biomolecules and surfaces is well-known in the scientific literature (Bentzen, et al., 2005; Charles, et al., 2009; Lakshmipriya, et al., 2013; Tomlinson, et al., 2006). Also, modification of biomolecules with dPEG® products increases the hydrodynamic volume of the conjugates. Increased hydrodynamic volume has distinct, recognized benefits for in vivo applications.


Biotinylation with dPEG® Products Allows Fine-Tuning of the Degree of Labeling of Biomolecules

As noted above, biotinylation with biotin alone or with LC-biotin limits the number of biotins that can be added to a protein. Moreover, even with low biotin loading of a protein, some disadvantages arise from the use of hydrophobic spacers with biotin. However, with Quanta BioDesign's dPEG® products, these disadvantages disappear. This is because water soluble dPEG® products eliminate aggregation and precipitation of proteins due to hydrophobic interactions.

Because biotinylation with dPEG® products does not force our customers to limit themselves to 2 – 3 biotins per protein molecule, biotinylation of free amines is limited by the number of surface accessible amines on the protein while maintaining binding capacity. Adding too many Biotin-dPEG® labels to a protein can interfere with the binding or activity of the protein. Nevertheless, Quanta BioDesign's Biotin-dPEG® products now give our customers freedom to tune the number of biotin labels that they put on a protein. This is a notable advancement in protein biotinylation. Indeed, in our in-house, unpublished work, we have labeled secondary antibodies with up to 14 biotin labels per protein molecule and have not observed visible aggregation or precipitation of antibody.

For more general information on dPEG® products see our What is dPEG®? and Frequently Asked Questions pages.


A Range of Biotinylation Products

Quanta BioDesign offers numerous biotinylation reagents for the effective biotinylation of biomolecules. Various functional groups for conjugation to free amines, thiols, carbonyls, and carboxylic acids are available. These will be discussed below.


Biotinylation to Free Amines with dPEG® Products

The most popular method for biotinylation of proteins and peptides is conjugation of biotin to free amines. This can be accomplished by direct coupling of biotin or biotin plus a spacer molecule to the target molecule using a carbodiimide such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, also known as EDC. More commonly, though, the carboxylic acid end of biotin or a biotin conjugate is activated with N-hydroxysuccinimide (NHS) and then reacted with free amines. Unlike direct conjugation with EDC, this method reacts with free, surface accessible amine groups on the target molecule without the risk of crosslinking free acid groups on one target molecule with free amine groups on another target molecule.

One limitation of conjugation to free amines is that not all amines on a large biomolecule (e.g., a protein) are accessible. For example, as noted in Avidin-Biotin Chemistry: A Handbook, bovine serum albumin has 59 lysine residues in its primary sequence, but only 30 – 35 of these lysines are available for biotinylation (Savage et al., page 34). Another limitation of this conjugation process is that the distribution of biotinylated amine groups is essentially random. If site-specific conjugation is needed, a different conjugation method will be required.


Site-Specific and Site-Controlled Biotinylation with dPEG® Products

For site specific biotinylation of biomolecules, Quanta BioDesign offers the ability to conjugate Biotin-dPEG® products to thiols using a maleimide functional group. Also, biotinylation of aldehydes and ketones — which can be found on, or introduced into, carbohydrate groups — can be accomplished with our oxyamine functionalized biotinylation products. To biotinylate surface accessible carboxylic acids (i.e., side chains of Glutamate and Aspartate and C-terminus amino acids) we offer Biotin-dPEG®n-amine products and product number 10219, Biotin-dPEG®4-hydrazide. The hydrazide will react with carboxylic acids to form a hydrazone, which can be cleaved at low pH for use in drug delivery.


Biotin modification of Gold and Silver Surfaces

For biotinylation of gold surfaces such as gold nanoparticles and quantum dots, Quanta BioDesign, Ltd., has Biotin-dPEG® products functionalized with lipoic acid. Lipoic acid is well known for forming two stable dative bonds with gold and silver, making it superior to thiol products for modifying gold surfaces. As such, these biotinylation products can be used in applications designed to capture biotin-binding proteins and peptides by affinity.


Biotinylation of Biomolecules with NHS-dPEG®n-Biotin and Biotin-dPEG®n-TFP ester

Quanta BioDesign, Ltd. offers two classes of products for biotinylation of free amines on biomolecules and surfaces. The first class is the NHS-dPEG®n-Biotin line of products. See Figure 3. These are Biotin-dPEG® acid products that have been activated with NHS. They are designed as direct replacements for traditional, hydrophobic LC-Biotin products.

Figure 3: Quanta BioDesign's NHS-dPEG®n-Biotin line of biotinylation products. The products are functionalized with biotin through an amide bond on one end of a discrete PEG (dPEG®) spacer consisting of 4, 12, or 24 ethylene oxide units, and with N-hydroxysuccinimide (NHS) on the opposite end. The NHS is joined as an ester to a propanoic acid group.
Figure 3: Quanta BioDesign's NHS-dPEG®n-Biotin line of biotinylation products.

These products offer clear improvements over biotinylation with biotin alone or with LC-Biotin products (see Figure 2, above). The principal improvement is that these products are water soluble. In addition, the spacers in these products are significantly longer than the aminocaproic acid spacer used in LC-Biotin products. This makes it easier for the terminal biotin moiety to bind in a (strept)avidin binding pocket. Moreover, as discussed above, these products convey the benefits of dPEG®, which include (in addition to water solubility) reduction of non-specific binding, elimination of aggregation and precipitation of protein, and increasing hydrodynamic volume. Furthermore, these products use familiar chemistry (the NHS group) for reacting with free amines.


Problems with NHS Esters

But NHS esters have shortcomings. Primarily, they hydrolyze relatively easily in water. The half-life of dissolved NHS ester in aqueous buffer decreases rapidly as the pH increases. In fact, above pH 8.0, the half-life of an NHS ester in solution is measured in minutes.

Tetrafluorophenyl Esters are a Superior Alternative to NHS Esters

As an alternative to NHS esters, Quanta BioDesign developed and sells active esters based on 2,3,5,6-tetrafluorophenol. See Figure 4, below. The tetrafluorophenyl (TFP) esters of dPEG® products offer superior performance compared to NHS esters.

Figure 4: Quanta BioDesign's Biotin-dPEG®n-TFP ester line of biotinylation products. As discussed in the text, these products offer superior performance compared the comparable NHS esters.
Figure 4: Quanta BioDesign's Biotin-dPEG®n-TFP ester line of biotinylation products. As discussed in the text, these products offer superior performance compared the comparable NHS esters.

While retaining the benefits of the NHS esters described above, TFP esters have improved hydrolytic stability compared to NHS esters. Also, TFP esters react with greater specificity towards free amines. In internal testing and comparison of TFP esters and NHS esters, our scientists consistently get obtain greater degrees of labeling with TFP esters than with NHS esters using optimized conditions for each. A previous internal study established the superiority of TFP esters over NHS esters. For these reasons, Quanta BioDesign strongly recommends that our customers switch from NHS esters to TFP esters if possible. For those customers who are unable to switch, however, we continue to provide the NHS esters.



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