antibody structure

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Peptide Antigen DesignEdit

Peptides are complex biomolecules that have unique chemical and physical properties. These properties are the direct result of their amino acid composition. Peptides can be designed de novo or based on peptide sequences from native proteins, depending on the desired application. In order to maximize the probability that antibodies against a synthesized peptide will recognize native proteins in target assays, antigen design is the key to choosing a suitable peptide sequence. Here, we have outlined several key principles that should be considered in the peptide design process. If you would like LifeTein to help you design your peptide, our technical support department can assist you.

In general, a good peptide antigen is derived from a sequence on the surface of the native protein, has a flexible structure (usually a loop), is a unique sequence, is easy to synthesize, and contains no post-translational modification sites.

Homology Considerations: There are two basic strategies that can be used to address peptide antigen design. Both approaches should be taken into consideration when analyzing the protein sequence.

  1. Choose a unique sequence that would help ensure specificity to the target protein.
  2. Choose a homologous peptide sequence that would allow a single antibody to recognize multiple proteins.

Epitope Selection: In general, most ideal antigenic epitopes are hydrophilic, surface-orientated, and flexible. This is because, in most natural environments, hydrophilic regions tend to be found on the surfaces of proteins and hydrophobic regions tend to be found hidden in the interior. Similarly, antibodies can only bind to epitopes on the surfaces of proteins and tend to bind with higher affinity when those epitopes are flexible enough to move into accessible positions.

Continuous versus Discontinuous Epitopes:

Protein structures determined using X-ray crystallography can help researchers conceptualize macromolecular protein complexes, visualize the impact of mutations, and even design targeted drug therapies. However proteins are not as rigid as their structural depictions imply. Inside cells, proteins fold and unfold constantly. Most antibodies target continuous epitopes, which are continuous sequences of amino acids. Antibodies will bind to these regions with high affinity if those sequences are not located within the protein interior. In some cases, antibodies can be generated against discontinuous epitopes, if those epitopes correspond to a fold in a peptide sequence or a place where two separate peptide chains meet. However this requires that the peptide used for immunization have a secondary structure similar to the epitope.


Peptide Antigen Design

If the research application in question is focused on specific protein domains, such as the C and N-terminals, or a particular state of the protein, such as phosphorylation, then peptide antigens and the corresponding antibodies against those antigens are relatively easy to use. However, protein conformation may interfere with antibody access to the target epitopes. The problem in this case is that a particular sequence may become inaccessible if it is hidden within the interior of the folded protein. It is important to consider this aspect of peptide design for antibody recognition purposes.

Targeting the N-terminus or C-terminus: To counter the risk of selecting an epitope buried within the protein, we normally recommend that antibodies be generated against the C-terminus or N-terminus, because these are often exposed. However, it is important to note that the transmembrane domain or C-terminus sequences of many membrane proteins are often too hydrophobic to act as antigens.


Peptide Antigen Design Sequence Selection Guideline

Sequence Length: In general, we recommend peptide sequences of 8–20 amino acids for antibody preparation. If the peptide is too short, it could not be specific enough for the antibodies to recognize the native protein with sufficient affinity. If the sequence is over 20 amino acids in length, it might lose its specificity and induce a secondary reaction.

Peptide Purity: For antibody generation and testing, peptide purity > 75% is usually sufficient. However, for biological activity studies, peptide purity > 95% is recommended.

Peptide Solubility Peptide solubility is strongly influenced by amino acid composition. It is advisable to keep the hydrophobic amino acid content below 50% and to make sure that there is at least one charged residue for every five amino acids.


Hydrophobicity and Hydrophilicity Example of a Peptide

Amino acid classifications Hydrophobic (non-polar): Ala, Ile, Leu, Met, Phe, Trp, Val Uncharged (polar): Asn, Cys, Gly, Gln, Pro, Ser, Thr, Tyr Acidic (polar): Asp, Glu Basic (polar): His, Lys, Arg

Peptides with a large numbers of hydrophobic residues, such as Leu, Val, Ile, Met, Phe, and Trp, have only limited solubility in aqueous solutions, and some are completely insoluble. Such peptides are usually difficult to use in experiments and can be difficult to purify. At physiological pH, Asp, Glu, Lys, and Arg all have charged side chains.

Some hydrophobic amino acids at nonessential positions can be substituted with conservative amino acids such as alanine or glycine, deleted, or replaced with analogues. A single conservative replacement, or the addition of a set of polar residues to the N- or C-terminus, may improve solubility.

Certain basic characteristics can be used to predict solubility:

  • Peptides that are shorter than 5 amino acids are usually soluble in aqueous solutions. If the entire sequence consists of hydrophobic amino acids, it will have limited solubility or may even be completely insoluble.
  • Hydrophilic peptides containing >25% charged residues (E, D, K, R, and H) and <25% hydrophobic amino acids are usually soluble in aqueous solutions.
  • Hydrophobic peptides containing 50% or more hydrophobic residues may be insoluble or only partly soluble in aqueous solutions. It is better to dissolve these peptides in organic solvents such as dimethylsulfoxide (DMSO) if they do not contain C, W or M, dimethylformamide (DMF), acetonitrile, isopropyl alcohol, ethanol, acetic acid, 4–8 M guanidine hydrochloride (GdnHCl), or urea prior to a careful dilution in aqueous solution.
  • Hydrophobic peptides containing >75% hydrophobic residues generally do not dissolve in aqueous solutions. Very strong solvents such as TFA and formic acid are required for the initial solubilization. The peptide may precipitate when added to an aqueous buffered solution. High concentration of organic solvent or denaturant may be required to dissolve these peptides.
  • Peptides containing a very high proportion (>75%) of D, E, H, K, N, Q, R, S, T, or Y are capable of building intermolecular hydrogen bonds (cross-links) and can thus form gels in concentrated aqueous solutions. These peptides should be dissolved in organic solvents. The initial solvent of choice should be compatible with the experiment. After dissolving the peptides in organic solvent, slowly add (dropwise) the solution to a stirred aqueous buffer solution. If the resulting peptide solution begin to show turbidity, you have reached the limit of solubility.

Difficult Amino Acids

  • Cysteine (C) and methionine (M)residues can undergo rapid oxidation, which can negatively influence the cleavage of protecting groups during synthesis and subsequent peptide purification. Methionine can be irreversibly oxidized to form methionine sulfoxide, which then forms methionine sulfone.Oxidation of cysteine occurs more quickly at higher pH. The thiol group in the peptide is easily deprotonated and readily forms intra-chain and inter-chain disulfide bonds. Whenever possible, sequences should be designed to have as few of these residues as possible. Conservative replacements can be made for some residues. For instance, cysteine can be replaced with serine and methionine with norleucine (Nle).When choosing a peptide from a native sequence, separating two Cys residues into two peptides will allow for better synthesis and a purer final product. This is because, when multiple cysteines exist on the same peptide, they are likely to form disulfide linkages. To prevent this, either a reducing agent such as dithiothreitol (DTT), or tris(2-carboxyethylphosphine) hydrochloride (TCEP) should be added to the buffer or the cysteines should be replaced with serine residues. Cysteine residues can affect the activity of the antibody. Because free cysteines are rare in vivo, they may not be recognized as part of the native peptide structure.
  • N-terminal glutamine (Q) is unstable. Glutamine at the N terminus can form cyclic pyroglutamate under acidic conditions during protecting group cleavage. Acetylating the N-terminal glutamine or substituting glutamine with pre-formed pyroglutamic acid or a conservative amino acid can prevent this.
  • N-terminal asparagine (R) should be avoided. The asparagine N-terminal protecting group can be difficult to remove during cleavage. The N-terminal asparagine should be removed or replaced with another N-terminal amino acid.
  • Aspartic acid (D) is very susceptible to dehydration and can form a cyclic imide intermediate. When paired with glycine, serine, or proline, the cyclic imide intermediate can be hydrolyzed and cause peptide cleavage under acidic conditions. Eventually, all of the aspartate on a given compound can be completely converted into the iso-aspartate analog. Avoid these combinations wherever possible by substitution or by shifting the sequence.
  • Multiple serine (S) or proline (P) residues can cause significant deletions during synthesis. This is especially true of proline residues, which can undergo cis/trans isomerization and reduce peptide purity.
  • Any series of glutamine, isoleucine, leucine, phenylalanine, threonine, tyrosine or valine can cause β sheets to form. These can prevent the growing peptide from dissolving completely during synthesis, causing deletion sequences. Avoid multiple and adjacent residues of above amino acids if possible. If such sequences cannot be avoided, it the β sheets patterns can be broken up by conservative substitution of Asn for Gln or Ser for Thr, by adding a Gly or Pro every third amino acid, or by shifting the sequence.
  • Glycine (G) in the third position from the N-terminus, especially if Pro or Gly is in position 1 or 2, can form diketopiperazine and pyroglutamic acid. Avoid the combination whenever possible.

Carrier Protein Coupling Considerations: Although coupling strategies can vary widely depending upon the sequence, it is important to remember that the peptide should be linked to the carrier protein via the carboxyl- or amino-terminal residue. If no internal cysteines are present, then a cysteine should be added to the sequence. Usually, we recommend conjugation with carrier protein to the N terminus of the peptide.

Peptides alone are usually too small to elicit an immune response sufficient to generate antibodies. This is why peptides of interest are conjugated to carrier proteins containing many epitopes that stimulate T-helper cells. These induce the B-cell response that generates the antibodies. The immune system reacts to the peptide-carrier complex as if it were a whole protein. Some of the antibodies so elicited target the linker region and the carrier protein itself in addition to the peptide of interest. These non-specific antibodies can be removed during the purification process.

Common carrier proteins used for antibody production:

  • KLH (keyhole limpet hemocyanin) is a copper-containing, non-heme protein found in arthropods and mollusks. It is isolated from Megathura crenulata and has a MW of 4.5 x 105–1.3 x 107 Da. KLH is the most commonly selected carrier because of its excellent immunogenicity.
  • BSA (bovine serum albumin), a plasma protein in cattle, is one of the most stable and soluble albumins. It has a MW of 67 x 103 Da and it contains 59 lysines. About 30–35 of these primary amines are accessible for linker conjugation. This makes BSA a popular carrier protein for weakly antigenic compounds.One disadvantage of BSA is that it is used in many experiments as a blocking buffer reagent. In such assays, antisera against peptide-BSA conjugates can generate false positives because these sera also contain antibodies against BSA.
  • OVA (ovalbumin), a protein isolated from hen egg whites, has a MW of 45 x 103 Da. It makes a good second carrier protein if antibodies are specific for the peptide.Thiol group modifications (via Cys side chains) are used for KLH, BSA, and OVA conjugation.

Carrier Protein for Antibody Generation

All modifications carrying thiol-reactive functional groups can be used. There are three commonly used reactive groups:

  • Iodoacetamides
  • Maleimides
  • Alkyl halides

Multiple antigenic peptides (MAPs) are artificially branched peptides. Lysine residues are used as a scaffolding core to form up to 8 branches with the same or different peptide sequences. MAP has a long history of use in immunological studies for antibody production. Some peptides render a lower immunological response. However, new structures, such as concentrated peptides in branched forms, can dramatically increase the immunological response.

Many antigenic peptides can be synthesized using standard SPPS. During this process, Boc-Lys(Boc)-OH is anchored to a resin. After treatment with TFA, cycles of deprotection and coupling are performed sequentially. The peptides intended for the immunological studies are then synthesized on each of the eight branches.

Branched proteins are unknown in nature. The synthesis of MAP is not generally straightforward. The space between the 8 branches can cause the peptides to aggregate on the resin, producing low coupling yields and deletions.

LifeTein uses PeptideSyn technology to overcome this problem through chemical ligation. This strategy provides the desired peptide dendrimer at higher yields than traditional methods. These branched structures then give the protein greater molecular weight, which is useful for immunogen purposes.

Mutiple Antigenic Peptides

Peptide Modifications

One great advantage of synthetic peptides that they can be made in the exact conformations or with the exact characteristics required for the application at hand. Peptides in general and specific amino acids have distinct moieties that are amenable to modification:

A. N-terminal amino group
B. C-terminal carboxyl group
C. ε-Amino group on lysine
D. Hydroxyl group on serine, threonine, and tyrosine
E. Guanidine group on arginine
F. Thiol group on cysteine


Peptide Modification Sites

While the list given above contains the most common modifications, it is by no means exhaustive. Many modifications are post-translational modifications and occur in vivo, while others take the form of substitutions of natural amino acids with non-natural variants. Additionally, tags or proteins can be chemically conjugated via cross-linking chemistry to the moieties listed above. Because of the C-to-N direction of synthesis, it is recommended that any tags or dyes be conjugated to the N-terminus so that only full-length peptides become labeled.

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