Biotinylation of Recombinant Proteins

Biotin (or vitamin B7) is an essential coenzyme that is required by all organisms for life. Indeed, biotin’s name literally means ‘life giving’. Biotin is covalently linked near the active sites of four major classes of carboxylase enzyme with a variety of metabolic roles. In most bacteria, plants, and fungi, biotin is synthesised by the organism, but in higher eukaryotes, biotin is sourced externally. Humans get biotin from a small number of foods as well as from gut bacteria.

In cells, biotin is added to proteins through enzymatic biotinylation, a process carried out by biotin protein ligases (BPLs). BPLs are enzymes of extraordinary specificity with most organisms having fewer than five enzymes that are biotinylated. For example, BirA, the BPL of E. coli, biotinylates only a single cellular protein; the BCCP subunit of acetyl-CoA carboxylase.

Avidin was discovered Esmond Emerson Snell after the observation that chicks fed on egg white were suffering from biotin deficiency. This was discovered to be due to an egg white glycoprotein sequestering biotin which was named Avidin^1,2. Avidin is now known to be produced by the oviducts of birds, reptiles, and amphibians. The exact purpose is unknown, but it is thought to inhibit bacterial growth by sequestering biotin. Streptavidin is a related protein discovered in the 1960s from the bacterium Streptomyces avidinii³, where it is also believed to act as an antibiotic factor. The key difference between these proteins is that unlike avidin, streptavidin and other bacterial derived avidins are non-glycoproteins.

The biotin-avidin interactions have evolved to be one of the strongest known non-covalent interactions in nature, with a K_d in the range of 10^-14 to 10^-15 M (picomolar to femtomolar range). The bond is resistant to denaturants, extreme pH, extreme temperature, and detergents. Both streptavidin and avidin bind with high affinity, however, avidin has a higher degree of non-specificity due to its higher pI and glycan modifications.

The strength of the biotin-avidin interaction makes it an attractive molecular tool. Biotin is small and unlikely to disturb the natural function of a molecule. Biotin can be added to molecules enzymatically or chemically to proteins and nucleic acids. In protein biology, biotinylation can be used for highly sensitive protein detection. Biotinylation can also be used for protein isolation, with the interaction strength giving stable immobilisation and allowing for strong wash conditions. This makes biotinylation a great tool for protein purification, HTS or biophysics.

Proteins can be specifically biotinylated using the natural specificity of the E. coli protein, BirA. BirA specifically biotinylates a single lysine (K122) on the BCCP protein⁴. Originally, BCCP was fused to proteins, but through peptide screening, a minimal peptide sequence was identified with a similar affinity to the natural substrate⁵. This was then further optimised to have an even higher affinity than BCCP⁶. Adding this AviTag peptide sequence to a protein allows specific biotinylation using recombinant BirA. We regularly use this technology at Peak Proteins to biotinylate proteins for our clients for a range of uses and also in collaboration with our parent company Sygnature Discovery for compound screening and SPR.

Biotinylation of recombinant proteins can also be carried out in vivo by co-expressing the target protein along with BirA. This can be done in all expression systems we offer at Peak Proteins; E. coli⁷, insect cells⁸, and mammalian cells⁹. We have been using in vivo biotinylation a lot recently at Peak Proteins because it can offer several advantages for our clients.

• Reduced handling time preventing loss of difficult to work with proteins
• Reduced processing times decreases costs
• More efficient biotinylation with the option to biotinylation in vivo and in vitro
• Reduced chance of BirA contamination

For an example of how we used in vivo biotinylation in insect cells for a client, please read out recent case study.

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