Protein biotinylation

In Rekom Biotech we have developed a product line of monobiotinylated proteins, offering some of the references of our catalog with a biotin in their C-terminus. This molecule allows the specific interaction of biotinylated proteins to streptavidin.

Our biotinylated proteins are bonded to a BCCP-tag in the C-terminus, with a lysine residue which is enzymatically biotinylated by the E. coli biotin ligase BirA. This single-point labelling technique has many advantages for commonly used binding assays:

  • The biotinylation only happens on the lysine residue of the BCCP tag.
  • There is NO interference with the target protein’s natural binding activities.
  • The protein orientation is uniform when immobilized on a streptavidin-coated surface such as nanoparticles.

Take a look at our monobiotinylated proteins and do not hesitate to contact us if you need anything.

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DISEASE/MICROORGANISM NAME REFERENCES DESCRIPTION DETAILS

Acquired immunodeficiency syndrome (AIDS) / Human immunodeficiency virus (HIV)

p24
RAG0057BIOT (biotinylated)
Viral capsid antigen

Babesiosis (or piroplasmosis) / Babesia canis

BcMSA1
RAG0020BIOT (biotinylated)
Merozoite Surface Antigen

Chagas / Trypanosoma cruzi

ChimChagas3
RAG0096BIOT (biotinylated)
Recombinant chimeric antigen for Chagas detection

Cytomegalovirus infection (CMV)

ChimCMV1
RAG0109BIOT (biotinylated)
Recombinant chimeric antigen which contains several antigenic determinants for CMV
ChimCMV2
RAG0110BIOT (biotinylated)
Recombinant chimeric antigen for CMV detection
pp52
RAG0090BIOT (biotinylated)
DNA polymerase processivity subunit

Epstein-Barr virus infection (EBV)

p18
RAG0049BIOT (biotinylated)
Viral capsid antigen

Leishmaniasis / Leishmania infantum

K39
RAG0061BIOT (biotinylated)
Parasite kinesin-related antigen

Neosporosis / Neospora caninum

NcGRA7
RAG0024BIOT (biotinylated)
Neospora caninum dense granule antigen GRA7

Phleum pratense (Timothy grass pollen)

Phl p 5a
RAL0003BIOT (biotinylated)
Unknown

Syphilis / Treponema pallidum

ChimSyphilis1
RAG0046BIOT (biotinylated)
Recombinant chimeric antigen for Treponema pallidum
ChimSyphilis2
RAG0064BIOT (biotinylated)
Recombinant chimeric antigen for Treponema pallidum
Tpp15
RAG0009BIOT (biotinylated)
Membrane lipoprotein
Tpp17
RAG0008BIOT (biotinylated)
Membrane lipoprotein
Tpp47
RAG0010BIOT (biotinylated)
Membrane lipoprotein

Triticum aestivum (Wheat)

Tri a 19
RAL0053BIOT (biotinylated)
Omega-5 gliadin, seed storage protein

Conventional chemical conjugation

Biotinylated protein
Figure 1. Biotinylated protein

In many ways, a conjugated protein can be the solution to different problems arisen in a new IVD test development. A common problem caused by most of the surfaces is protein denaturation due to the relatively high surface by hydrophobicity. Additionally, there may be larger steric influence on binding events due to the close proximity of the surface and the sensor molecules. The specific orientation may also improve the stability of attached proteins and increase the sensitivity of the assay by exposure of its antigenic regions.

Conventional conjugation methods generally work well with antibodies. However, results with many proteins of less well-established structures are less constant. This is probably the main reason why Double-Antigen Sandwich ELISAs (DAS) are not as commonly used for antibody detection as indirect ELISA.

To circumvent the effect on antigenic structure caused by conventional chemical conjugation that could result in decrease in sensitivity of DAS-ELISAs, the use of monobiotinylated proteins is perfect.

Why use our biotinylated proteins?

The extremely specific and high affinity binding of biotin by avidin and/or streptavidin (Kd ≈ 10-14M) results in specific detection systems of very high sensitivity. A clear advantage of this system is that with a common strep-HRP, we can obtain conjugated complex of all our references without the necessity of performing the peroxidation of each one.

The biotin is fused to a linker which maintains the molecule away to the protein surface, avoiding steric hindrance between the biotin and the antigenic regions involved in Ab-binding. Thus, the Ab interaction will not be compromised with the conjugation.

Interaction between an antigen vs Fab region of an Ab
Figure 2. Interaction between a protein (green) vs Fab region of an Ab (pink). In red are specified the amino acids that usually are used in conjugation procedures. Some of these amino acids are part of the epitope region of the protein. The conjugation would interfere with the Ab-binding, decreasing the antigenic capacity of the protein.

As there is just one biotin per protein molecule, our conjugated proteins will show a higher inter-lot reproducibility and this will facilitate the reproducibility of the diagnostic tests developed with them.

Biotinylated proteins applications

Protein orientation in streptavidin-coated plates
Protein orientation in streptavidin-coated plates
Detection in IgM capture assays
Detection in IgM capture assays
Detection in DAS assays
Detection in DAS assays
Binding to gold and other nanoparticles coated with streptavidin
Binding to gold and other nanoparticles coated with streptavidin