PROTEOMICS & GENOMICS/ GENETIC ENGINEERING - BIOTINYLATION

1. BARKATULLAH UNIVERSITY
Department of Biochemistry & Genetics
Paper code: E1 304: PROTEOMICS & GENOMICS
Unit: IV
Topic: BIOTINYLATION
BY
Ms. NIDHI PURANIK , PhD
Faculty Member
Department of Biochemistry & Genetics

2.  Biotinylation is the process of attaching biotin to proteins and other macromolecules.
 Biotinylation reagents are available for targeting specific functional groups or
residues, including primary amines, sulfhydryls, carboxyls and carbohydrates.
 Photoreactive biotin compounds that react nonspecifically upon exposure to
ultraviolet (UV) light are also available and expand the scope of the molecules that
may be biotinylated.
 The variety of biotinylation reagents with different functional group specificities is
extremely useful, allowing one to choose a reagent that does not inactivate the target
macromolecule.
BIOTINYLATION
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3.  Besides functional group specificity, biotinylation reagents are available with different
solubility characteristics to focus biotinylation to distinct microenvironments either
inside or outside cells.
 Cleavable or reversible biotinylation reagents enable the specific elution of
biotinylated molecules from biotin-binding proteins.
 The variability of these reagents substantially expand the range of applications for
avidin–biotin chemistry.
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4. CHEMICAL STRUCTURE OF BIOTIN
Biotin, also known as B-vitamin B7 (formerly vitamin H and coenzyme R) is water soluble.
The molecule is comprised of a ureido ring joined with a tetrahydrothiophene ring. A
coenzyme for carboxylase enzymes, biotin is required for the synthesis of fatty acids,
isoleucine and valine. Biotin is also involved in in gluconeogenesis.
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5. Biotin labeling
 Proteins that are biotin labeled (i.e., biotinylated) are routinely detected or purified with
avidin conjugates in many protein research applications, including the enzyme-linked
immunosorbant assay (ELISA), western blot analysis, immunohistochemistry (IHC),
immunoprecipitation (IP) and other methods of affinity purification, cell surface labeling
and flow cytometry/fluorescence-activated cell sorting (FACS).
 Besides a strong affinity for avidin, biotin exhibits two characteristics that make the
molecule ideal for labeling proteins and macromolecules. First, biotin is comparatively
smaller than globular proteins, which minimizes any significant interference in many
proteins and allows multiple biotin molecules to be conjugated to a single protein for
maximum detection by avidin.
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6.  Second, as shown in the diagram below, biotin has a valeric acid side chain that is
easily derivatized and conjugated to reactive moieties and chemical structures without
affecting its avidin-binding function. This feature allows many useful biotinylation
reagents to be created.
Comparison of biotin and biocytin. Biocytin differs from biotin by the addition of a
lysine group attached to the valeric acid side chain.
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7.  Biocytin is a derivative of biotin found in serum and urine that has an added
lysine group coupled at the ε-amino acid side chain to the valeric acid side
chain.
 As shown in above diagraam, biocytin is longer than biotin, which makes the
molecule useful in making long-chain biotinylation reagents. Biocytin can
also be used to make trifunctional crosslinking reagents because of the free
carboxylate group and α-amine.
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8.  Biotinylation, also called biotin labeling, is most commonly performed through
chemical means, although enzymatic methods are also available.
 Chemical methods provide greater flexibility in the type of biotinylation needed
than enzymatic approaches and can be performed both in vitro and in vivo.
 Enzymatic methods require the co-expression of bacterial biotin ligase and an
exogenously expressed protein of interest that is modified to carry a biotin
acceptor peptide, which provides a more uniform biotinylation than chemical
methods and can be cell compartment specific. Because of the greater
availability of chemical biotinylation reagents and customization, this is
commonly use in study.
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9. Determining the right biotinylation reagent to use for a specific application and
protein of interest depends on a number of factors that must be carefully considered
and optimized, including:
Solubility—Modifications in biotinylation reagent solubility allow access to target
proteins in hydrophobic or hydrophilic environments and influence the solubility of
the biotinylated protein.
Spacer arm length—The availability of biotin for avidin binding is influenced by
the length of the spacer arm.
Cleavability/reversibility—Captured biotinylated proteins can be recovered or
purified by cleaving the biotin molecule from the target protein or reversing
attenuated biotin–avidin interactions.
Functional group—Specific reactive moieties bind to any amino acids or to the
functional groups of specific amino acids for nonselective or targeted biotinylation,
respectively. 9

10. Biotinylation reagent solubility
• Biotinylation reagent solubility is based on the solubility of the reactive moiety, the
spacer arm or a combination of both.
• Some reactive groups are inherently charged and are therefore water-soluble, while
uncharged groups require modification (e.g., sulfonation; see section on NHS esters
below).
• A common method to make the spacer arms soluble is to incorporate poly (ethylene
glycol), or PEG, which is highly soluble and flexible.
• Spacer arms comprised of PEG can make biotinylation reagents with uncharged
reactive groups soluble or those with charged reactive groups more soluble.
• Additionally, the increased solubility stemming from PEG-containing biotin tags helps
prevent biotinylated protein aggregation during long-term storage compared to non-
biotinylated proteins.
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11. Poly(ethylene glycol) increases the solubility of biotinylation reagents.
A four-ethylene glycol chain (PEG4) was conjugated onto the spacer arm between
the reactive moiety (red) and biotin (blue).
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12. Spacer arm length
• The ability of avidin to bind to biotin molecules on biotinylated proteins is dependent
upon the availability of biotin without steric hindrance from multiple biotins on the
same protein.
• Longer spacer arms can enhance the detection sensitivity of the target protein, because
more biotin molecules are available for reporter-conjugated avidin binding.
• For biotinylation, the spacer arm is defined as the distance from the end of the
conjugated amino acid to the end of the biotin molecule. Thus, the spacer arm length
of a biotinylation reagent can be the difference between strong protein
detection/purification and weak or no detection/purification because of the availability
or unavailability of biotin for binding to avidin conjugates, respectively.
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13. Examples of variable spacer arm lengths.
Chemical groups (black) modify the distance between the reactive moiety (red) and biotin
(blue) to regulate the length of the spacer arm. The reagents shown are (A) EZ-Link NHS-
Biotin, (B) NHS-LC-Biotin and (C) Sulfo-NHS-LC-LC-Biotin. 13

14. Cleavable biotin labeling reagents
• Cleavable biotinylation reagents allow the purification of biotinylated proteins after
capture or the removal of avidin-conjugated protein from a biotinylated sample.
• As shown below, cleavable biotinylation reagents are designed with disulfide bonds in
the spacer arm.
• Under reducing conditions (50 mM dithiothreitol, 10 mM 2-mercaptoethanol or 1%
sodium borohydride), the disulfide bonds are cleaved, releasing the biotin tag and any
avidin conjugate bound to it.
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15. Cleavable biotinylation reagents.
These reagents are designed with a disulfide bond that is cleaved under reducing
conditions (dotted line) to release the biotin tag (blue) from the tagged protein
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16. Functional group targeting
The goal of biotinylation is to label a protein of interest in such a way that the normal
biological function of the protein is not significantly interrupted. Even though biotin is
small, biotinylation can interfere with normal protein function if the biotinylation reagent is
conjugated to amino acids that regulate protein activity such as binding to substrates. Many
different reactive moieties are available to reduce potential interference by targeting
different specific amino acid functional groups, including:
Primary amines (-NH2)—This group is located at the N-terminus of each polypeptide
chain and in the side chain of lysine (Lys, K) residues.
Sulfhydryls (-SH)—This group is located in the side chain of cysteine (Cys, C). Often, as
part of a protein's secondary or tertiary structure, cysteines are joined together via disulfide
bonds (–S–S–) between their side chains.
Carboxyls (-COOH)—This group is located at the C-terminus of each polypeptide chain
and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E).
Carbonyls (-CHO)—This aldehyde group can be created by oxidizing carbohydrate groups
in glycoproteins. 16

17. Avidin–biotin interaction
• The interaction between biotin (vitamin H) and avidin is a useful tool in
nonradioactive methods of purification, detection, immobilization, labeling, viral
vector-targeting and drug targeting systems.
• The extraordinary affinity of avidin for biotin is one the strongest known non-covalent
interactions of a protein and ligand (Ka=1015M-1) and allows biotin-containing
molecules in a complex mixture to be discretely bound with avidin conjugates.
• The bond formation between biotin and avidin is very rapid, and once formed, it is
unaffected by extremes in pH, temperature, organic solvents and other denaturing
agents.
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18. Application
1. Purification, 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.
2. 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).
3. Detection, 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.
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19. 4. 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.
5. 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.
6. 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.
7. Furthermore, biotinylation of MHC molecules to create MHC multimers has become
a useful tool for identifying and isolating antigen-specific T-cell populations.
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