Keyhole limpet hemocyanin

Keyhole limpet hemocyanin (KLH) is a large, multisubunit, oxygen-carrying, metalloprotein found in the hemolymph of the giant keyhole limpet Megathura crenulata that lives off the coast of California from Monterey Bay to Isla Asuncion off Baja California[1].

Protein properties

Keyhole limpet hemocyanin is an extremely large, heterogeneous protein consisting of subunits with a molecular weight of 350,000 and 390,000 in aggregates with molecular weights of 4,500,000-13,000,000. Each domain of a KLH subunit contains two copper atoms that together bind a single oxygen molecule (O₂). When oxygen is bound to hemocyanin, the molecule takes on a distinctive transparent, opalescent blue color.

Purification

KLH is purified from the hemolymph of Megathura crenulata by a series of steps that typically includes ammonium sulfate precipitation and dialysis, and may involve chromatographic purification to obtain the highest purity. KLH purification may also include endotoxin removal, but this step is often unnecessary because the endotoxin serves as an adjuvant when injected for antibody production.

If the protein becomes denatured or if the copper ions are lost in the purification process, the opalescent blue color disappears and the solution becomes a dull grayish color. Denaturation of KLH also results in a tendency of the protein to aggregate and precipitate from solution.

Use in biotechnology

Keyhole limpet hemocyanin is used extensively as a carrier protein in the production of antibodies for research, biotechnology and therapeutic applications. Haptens are substances with a low molecular weight such as peptides, small proteins and drug molecules that are generally not immunogenic and require the aid of a carrier protein to stimulate a response from the immune system in the form of antibody production. KLH is the most widely employed carrier proteins for this purpose. KLH is an effective carrier protein for several reasons. It's large size and distinct/foreign epitopes generate a substantial immune response, and abundance of lysine residues for coupling haptens, allows a high hapten:carrier protein ratio increasing the likelihood of generating hapten-specific antibodies.

KLH may also be a challenging molecule to work with because of its propensity to aggregate and precipitate. Aggregates remain immunogenic, but limit the ability to conjugate haptens and are difficult to manipulate in the laboratory. A high quality KLH preparation with the clear opalescent blue color is the best indicator of KLH solubility.

Hapten coupling

Haptens can be coupled to KLH using several methods. A simple one-step coupling can be performed using the crosslinker EDC to covalently attach carboxyls to primary amines. This method is the simplest to perform and the "random" orientation allows for antibody generation against all possible epitopes, but it generally results in some degree of polymerization, which decreases solubility making the conjugate more difficult to handle.

KLH can be activated with the crosslinker Sulfo-SMCC, which converts lysine residues to sulfhydryl-reactive maleimide groups. A sulfhydryl-containing hapten can then be reacted with the KLH to complete the immunogen without causing polymerization. The specificity of this reaction is ideal for situations where the cysteine is located away from the desired epitope (e.g. in peptides where a terminal cysteine can be added to either end of the peptide). Maleimide activated KLH, where the first part of this two step procedure has been completed, is commercially available.

Other carrier proteins

• Concholepas concholepas hemocyanin (marketed as Blue Carrier®)
• Bovine serum albumin (BSA)
• Cationized BSA (cBSA)
• Ovalbumin