Control of protein orientation for enhanced compound detection

Fusion protein presents better binding sites and improved toxin detection

Medical & Biotechnology Sensors

Ricin structure.

Many immunoassays rely on monoclonal or polyclonal antibodies (such as Immunoglobulin G) derived from mice, rabbits, goats, or sheep as recognition elements. Considerable work has demonstrated that oriented capture molecules retain their binding ability and improve the performance of these assays.

However natural proteins have limited means by which they can be oriented.

In many respects it can be even more critical to orient small recombinant antibody derivatives because they have been reduced to only their active domain, thus reducing the number of random attachment sites. The same may be true of many recombinant enzymes as well.

While there have been specialized methods developed for particular surfaces or using non-natural amino acids, the vast majority of covalent protein surface immobilization, as well as dye molecule attachment chemistry, make use of either a carbodiimide or succinimide ester chemistry, which links a carboxyl group on one molecule to an amine on another. The limitation for successful orientation of proteins with these chemistries is that proteins possess a number of carboxyl and amine residues spread across their surface. The challenge has been to successfully orient the protein or attach the dye (or biotin) in a particular location to preserve the maximal activity of the protein.

As a solution to the above, scientists at the Navy Research Lab have developed a small positively charged peptide termed sdAb-GS-3K which provides oriented immobilization of an anti-ricin sdAb and improvement in the ability of a sensor to detect ricin. Tests show the oriented immobilized sdAb-GS-3K to provide enhanced detection of ricin relative to the randomly attached sdAb.

One key advantage of this methodology is that it makes use of the surface chemistry already being widely utilized, thus it is relatively transparent to the user. Many detect and measure systems make use of carboxyl-modified surfaces for the attachment of biomolecules and the technique described here makes use of these same surfaces but achieves a much more active and oriented molecule on that surface.

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