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KLH (Keyhole Limpet Hemocyanin) peptide conjugation service

SB-PEPTIDE offers to conjugate peptides to KLH (Keyhole Limpet Hemocyanin) for immunology applications (antibody generation…).

Keyhole Limpet Hemocyanin

Hemocyanin are large metallo-glyco-proteins that transport oxygen in bodies. Keyhole limpet hemocyanin (KLH) is an the hemocyanin from the giant keyhole limpet, Megathura crenulata, a species of keyhole limpet that naturally lives off the coast of California. KLH is a multi-subunit complex protein containing glycosylations and about 3400 amino acids for a total molecular weight above 400 kDa. The KLH oligomerise to form a larger complex of 20 monomers. There are two existing KLH proteins: KLH-1, uniprot Q10583 and KLH-2, uniprot Q10584. 

KLH contains primary amines (around 150) and carboxylic residues (around 500) which are used as conjugation sites for attaching haptens.

KLH in solution is naturally aggregating giving a cloudy apparence. Neverthless it is not impacting its immunogenicity.

KLH as a carrier protein

KLH is widely used as a carrier protein in immunology to improve immunogenicity of haptens (peptides, antibodies, glycans, lipids…) in order to generate antibodies as well as for some vaccine applications. The reason for this benefit is not fully determined. It is probably due to its phylogenetically distance with mammalians combined with its large size similar to microbes which can be easily detected by the immune system. Then antigen-presenting cells will process the KLH-hapten complex and present KLH fragments as well as conjagted haptens. This is allowing to improve the presentation of haptens that are not always detected or rapidly cleared from the body due to small size.

Peptide-KLH conjugation strategies

The usual approach to conjugate a peptide to KLH is to attach it via one of its extrimity (C-ter or N-ter). Once defined, the peptide sequence will be important to determine the conjugation strategy. Indeed, peptides can be conjugated to KLH via their free amines, free carboxyls or free thiols. Depending on which one is available and where in the sequence, the KLH conjugation strategy will be defined.

The most convenient and universal strategy is the use of thiol-containing peptides to conjugate them to maleimide-activated KLH. A thiol function can be introduced in peptide synthesis by the addition of a cysteine in C-ter or N-ter. In case a cysteine is already present in the sequence, this approach is normally not adapted. Nevertheless, SB-PEPTIDE has developed specific strategies to circumvent this limitation by using a proprietary linker.

Alternatives can be the conjugation via free amines or carboxyls of peptides.

KLH conjugation service

SB-PEPTIDE can synthesize peptides and conjugate them to KLH (as well as BSA, OVA, CRM197…), from 1mg conjugated to much more. Conjugates can be prepared in solution in PBS or in powder if required.

KLH can not be analyzed by gel migration or mass spectrometry due to its large and complex structure. When conjugating thiol-containing peptides, remaining free thiols are quantified by an optical measurement method. For other conjugation strategy, no quality control can be performed. The most important is to rely on a highly experimented partner in KLH conjugation as SB-PEPTIDE who will ensure an appropriate conjugation.