Carrier conjugation

Conjugation to carrier proteins

Immunization with naked peptide antigens is very often not successful, as peptides are often too small to generate significant immune responses by themselves. In order to solve this problem, peptides should be conjugated to large carrier proteins, such as Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or Ovalbumin (OVA). To ensure the effective and successful generation of anti-peptide antibodies, the use of peptide – protein conjugates is always recommended.

Peptide – Carrier protein coupling step by step

1. Selecting the carrier protein

Researchers may select a carrier protein form a range of options, of which the most commonly used carrier proteins are the following:

  • KLH (Keyhole Limpet Hemocyanin, MW 4.5×10⁵ – 1.3×10⁷ Da) is the most commonly used carrier protein, most likely because it has a significantly higher immunogenicity as compared to other proteins. KLH-conjugated peptides often have limited solubility in water, which results in a cloudy appearance. The turbidity does not affect its immunogenicity and the resulting solution can be used for successful immunizations.
  • BSA (bovine serum albumin, MW 67×10³ Da) is one of the most stable and soluble albumins available. It contains 59 lysines, of which approx 30-35 are accessible for conjugation. Therefore BSA is a popular carrier protein for weakly antigenic compounds.
  • OVA (ovalbumin, MW 4.5×10⁴) is a protein isolated from chicken eggs. It is often used as a control carrier protein to verify that antibodies are specific for the target peptide rather than the carrier protein itself (e.g. KLH/BSA).

Conjugation to other carrier proteins (e.g. HSA, Thyroglobulin) is available upon request

2. Peptide design & synthesis

Depending on the linkage chemistry to be used, we synthesize your peptides with the appropriate chemical handles at the right position, such that the peptide gets optimally exposed at the surface of your protein carrier. If desired, we can also introduce a rigid or flexible spacer to optimally finetune the distance to the carrier surface.

3. Linkage Chemistry

In most cases we apply one of two different linkage chemistries for peptide-to-protein conjugation, i.e. the standard ‘maleimide-based’ technology or the more advanced ‘Hydralink’ technology.

The ‘classical-type’ linker connects to the protein via formation of a standard amide-bond to any free amine group (free lysine side chain or N-terminus), whereas the peptide gets connected to the linker via conjugate addition of a free thiol to a maleimide functionality. This provides chemically stable peptide-protein linkages that cannot be destroyed under standard laboratory conditions. There are various linkers available in this format that mainly differ in size (MCS, GMBS), rigidity (MCS vs. MBS), and water-solubility (MBS vs. sMBS).

Name Chemical structure Reactivity Å H₂O Solubility
N-(ε-Maleimidocaproyloxy) succinimide ester
amine + sulphydryl ~9.0 NO
sEMCS (= sulfoEMCS)
N-(ε-Maleimidocaproyloxy)sulfo succinimide ester
amine + sulphydryl ~9.0 YES
m-Maleimidobenzoyl-N-hydroxysuccinimide ester
amine + sulphydryl ~7.5 NO
sMBS (= sulfoMBS)
(m-Maleimidobenzoyl-N-hydroxy)sulfo succinimide ester
amine + sulphydryl ~7.5 YES
Maleimidobutyryloxy succinimide ester
amine + sulphydryl ~6.5 NO
sGMBS (= sulfoGMBS)
(Maleimidobutyryloxy)sulfo succinimide ester
amine + sulphydryl ~6.5 YES

Next to this, we also apply the ‘Hydralink’ technology that couples peptides with free aminoxy (RO-NH2) or hydrazino-nicotinamide (HyNic) groups to any desirable carrier protein. To this end, the carrier protein is first derivatized with the reagent ‘S-4FB’ or ‘sulfo-S-4FB’ that converts a free amino group at the protein surface into an benzaldehyde group, which reacts with the reactive aminoxy or HyNic groups on the peptide. The technology has an important advantage over the standard thiol-maleimide chemistry, because it can be quantified by UV-spectroscopy at 354 nm. Moreover, this chemistry leaves disulfide bonds untouched and completely intact and avoids the problem of SS-bond scrambling when using free thiols.

Name Chemical structure Reactivity Å H₂O Solubility
p-Formylbenzoyl-N-hydroxysuccinimide ester
amine + aminoxy ~7.5 NO
(p-Formylbenzoyl-N-hydroxy) sulfo succinimide ester
amine + aminoxy ~7.5 YES

4. Quality control

We perform standard analyses to sufficiently characterize your peptide-protein conjugates for direct use. This involves a.o. SDS-PAGE to semi-quantify free BSA+carrier.

In addition to that, additional analyses can be performed as per customer requirements or needs. This includes HP-SEC analyses, to semi-quantify the amount of free peptide, or determination of the Peptide-Protein Ratio (i.e. the average peptide-loading).

Peptide-Protein Ratio is determined via amino-acid analysis. In order to obtain reliable data from these measurements, free peptide in the protein-peptide conjugate is removed upfront, e.g. via the ‘stirred-cell’ technique.

For conjugates in which the HydraLink technology has been applied, peptide-protein ratio can also be determined via UV-spectroscopy. The number of ‘free’ 4FB-units on the protein can be quantified via reaction with 2-hydrazinopyridine, a compound that absorbs at 350 nm. Determination of the amount of free 4FB before and after peptide-conjugation gives the average peptide/carrier ratio. These additional analyses are available upon customer request.

5. Delivery

The conjugates are supplied as chilled solutions since it has been shown that either reconstituted freeze dried materials or frozen solutions deliver sub-optimal immunization results.