CD Biosynsis offers premier BirA Enzyme-Mediated Biotinylation services, providing the highest level of site-specificity available for protein tagging. While chemical methods target broad functional groups like amines or thiols, BirA-mediated biotinylation utilizes the absolute substrate precision of the biotin ligase enzyme from Escherichia coli. By recognizing a specific 15-amino acid peptide sequence known as the AviTag or Acceptor Peptide (AP), BirA covalently attaches biotin to a single, predetermined lysine residue within that motif, ensuring 100 percent regioselectivity and a uniform 1:1 labeling ratio.
Our enzymatic platform is the ideal choice for applications requiring oriented protein immobilization, such as Surface Plasmon Resonance (SPR), Single-Molecule Studies, and the development of high-fidelity detection assays. Because the reaction occurs under physiological conditions and at a specific distal tag, the native structure and biological activity of your protein are perfectly preserved. Whether you require in vitro biotinylation of purified proteins or in vivo biotinylation within a cellular chassis, our expert team provides a complete solution from AviTag vector construction to the delivery of high-purity biotinylated conjugates.
Get a QuoteThe BirA enzyme is a 35 kDa bifunctional protein that acts as both a DNA-binding transcriptional repressor and a biotin-protein ligase. In nature, BirA specifically biotinylates the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. By engineering a minimal 15-residue recognition sequence (GLNDIFEAQKIEWHE) onto your target protein, we can hijack this natural system to achieve "surgical" biotinylation. This process is ATP-dependent and proceeds through a highly reactive biotinyl-5-adenylate intermediate, ensuring that only the specific lysine in the AviTag is modified.
The primary advantage of the BirA system is the homogeneity of the final product. Chemical biotinylation often creates a mixture of proteins with zero, one, two, or more biotin tags at various locations, which can lead to batch-to-batch inconsistency and activity loss. With BirA, every molecule in the population is biotinylated at exactly the same spot. This uniformity is crucial for quantitative assays and structural biology, as it allows for the precise orientation of the protein when captured on streptavidin-coated surfaces, ensuring that the active site is always facing the mobile phase.
Mechanism
Addition of high-purity recombinant BirA enzyme, D-Biotin, and ATP/Mg2+ to a purified AviTagged protein in a controlled buffer environment.
Control
Allows for the highest degree of reaction monitoring and post-conjugation purification to ensure 100 percent labeling efficiency.
Method
Simultaneous expression of the AviTagged target protein and the BirA enzyme within the host cell (E. coli, yeast, or mammalian cells).
Advantage
The protein is biotinylated as it is synthesized, eliminating the need for an additional in vitro enzymatic step and simplifying the purification pipeline.
N vs. C Terminus
The AviTag can be placed at either terminus or within internal flexible loops, depending on which orientation provides the best functional exposure.
Cleavability
Integration of protease cleavage sites (e.g., TEV or Thrombin) between the protein and the AviTag for optional tag removal after purification.
Our systematic pipeline ensures high-yield biotinylation with exhaustive analytical verification of the site-specific tag.
1. Vector Design & Expression
2. Enzymatic Reaction
3. High-Res Purification
4. Efficiency Verification
Subcloning the target gene into an AviTag-containing vector. Optimization of expression conditions to ensure high solubility of the tagged protein.
In vitro incubation with recombinant BirA, Biotin, and ATP. For in vivo projects, we utilize specialized E. coli strains (e.g., AVB101) that constitutively express BirA.
Confirmation of 1:1 biotinylation using a Gel-Shift Assay (with Streptavidin) or high-resolution ESI-MS mass spectrometry. Verification of biological activity via functional assays.
Absolute Site-Specificity
Biotinylation occurs only at the specific lysine within the AviTag, preventing any interference with the protein native lysines.
Preserved Potency
The mild enzymatic reaction conditions and distal tag placement ensure that the protein native conformation and activity remain 100 percent intact.
Uniform Orientation
Standardized tagging allows for uniform orientation during surface immobilization, critical for high-quality SPR and biosensor data.
Comprehensive Analytics
Full validation of biotinylation efficiency using mass spectrometry, ensuring every batch meets the highest standards of homogeneity.
1. What is the AviTag sequence?
The AviTag is a 15-amino acid peptide (GLNDIFEAQKIEWHE) that is specifically recognized by the E. coli BirA biotin ligase. It is significantly smaller than many other affinity tags, minimizing its impact on protein folding.
2. Can I use BirA to biotinylate a protein without a tag?
No. BirA is highly specific for the AviTag (or the natural BCCP subunit). For proteins without this specific sequence, chemical biotinylation methods must be used.
3. How do you verify that the biotinylation is 100 percent complete?
We utilize a Streptavidin-induced Gel Shift Assay, where the biotinylated protein shows a clear mass increase upon binding streptavidin. For absolute quantification, we use high-resolution Mass Spectrometry.
4. Is the AviTag better at the N-terminus or C-terminus?
It depends on the protein structure. We perform structural modeling to ensure the tag is surface-accessible and distal to the active site to prevent any loss of function.
5. Does the BirA enzyme remain in the final sample?
No. We employ purification steps (such as SEC or affinity chromatography) to ensure that the BirA enzyme, unreacted biotin, and ATP are completely removed from the final biotinylated protein.
6. Can I biotinylate proteins in mammalian cells using BirA?
Yes, by co-expressing a secretion-targeted or cytosol-targeted BirA enzyme along with your AviTagged protein, we can achieve high-efficiency in vivo biotinylation in mammalian systems.
7. Is the BirA reaction reversible?
The covalent bond formed between biotin and the lysine residue is permanent. However, if you use a cleavable linker between the protein and the AviTag, you can release the protein after capture.
8. What is the typical turnaround time for an AviTag project?
A full project, from gene synthesis and expression to purified biotinylated protein delivery, typically takes 6 to 10 weeks.
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The CRISPR gene editing system with its Cas9 version stands as a vital instrument for current biological research. CRISPR technology enables gene knockout (KO) through permanent gene expression blockage achieved by sequence disruption. Various scientific domains including disease modeling and drug screening employ this technology to study gene functions. CRISPR KO technology demonstrates high efficiency and precision but requires confirmation and verification post-implementation because unsatisfactory editing may produce off-target effects or incomplete gene knockouts which impact experimental result reliability. For precise and efficient Gene Editing Services - CD Biosynsis, Biosynsis offers comprehensive solutions tailored to your research needs.
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