When making peptide antigens, some researchers believe that one should block the C-terminal if the peptide comes from the N-terminal of the protein and that one should block the N- terminal if making a peptide that comes from the C-terminal of a protein. They believe that if the peptide comes from an internal region of the protein that it should have both terminals blocked. However, acetylation may not be necessary because the pKa of a peptide alpha-amino group is about 7.5 so the peptide is only about half protonated at neutral pH. Based upon the observations from many of our researchers, solubility should be your primary concern.
The following advice was given to readers of the ABRF bulletin board by Dr. John Stewart, Department of Biochemistry Univ. of Colorado Medical School, Denver, CO. He is the co-author along with Dr. Janice Young of SOLID PHASE PEPTIDE SYNTHESIS which is available from Pierce Chem. Co. SPPS is known to some as the "Bible of Peptide Synthesis" and worth a look by anyone interested in synthetic peptides.
To: Recipients of ABRF List:
To predict immunogenic sites in proteins, we use a "computerized manual" system:
1. Do a secondary structure prediction on the protein sequence. We like the Chou-Fasman program results. Particularly look for turns.
2. Calculate hydrophobicity, using a running average of 6 residues. Fauchere-Pliska or Hopp-Woods hydrophobicity data seem to give better results than Bull-Breese. Look for hydrophilic regions that also predict to be turns.
3. Predict glycosylation sites:
a. N-linked CHO chains occur at Asn-X-Thr or Asn-X-Ser sequences in turns.
b. O-linked CHO chains occur at Ser-X-X-Pro or Thr-X-X-Pro sequences in turns.
PREDICTION: The most hydrophilic site in the protein, if it is in a turn and NOT predicted to be glycosylated, will have a high probability of immunogenicity. For secondary sites apply the same rules to successively less hydrophilic regions.
We use internet sources like PROWL at Rockefeller University http://prowl.rockefeller.edu/ and ExPASy Molecular Biology Server http://us.expasy.org/ for determining the most immunogenic regions of a protein. You may wish to do an internet search using keywords such a "hydrophobicity and hydrophilicity" and "predict" to find newer or better programs.
The following is a summary of conjugation methods from the University of Michigan Protein and Carbohydrate Structure Facility by Dr. Phil Andrews from his WWW page.
Title: Conjugation of Peptide Antigens to Carriers
Peptide antigens are frequently coupled to high molecular weight carriers (usually proteins) prior to inoculation. Conjugation confers several advantages, including increased resistance to degradation,improved antigenicity, and enhancement of depot formation. The coupling method and the carrier used for conjugation are dependent on the chemical structure of the peptide, the species inoculated, and the use for the antiserum. Investigators should keep in mind that multiple antigenic peptides (MAPs) are an excellent alternative to classical peptide/protein conjugates for many applications.
|EDAC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide]||Sigma, etc|
|CHMC [1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate||Sigma, etc.|
|MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester)||Pierce, etc.|
|SMCC [Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carbonate]||Pierce, etc.|
|Table I. Summary of common methods for conjugation.|
|Carbodiimide||Glu/Asp to Lys/a-NH2||EDAC, CHMC, etc|
|Diazo (Benzidine)||Tyr/His/Lys to Tyr/His/Lys||bis-diazotized benzidine|
|Glutaraldehyde||Lys to Lys||Glutaraldehyde|
|Heterobifunctional||Cys to Lys||SMCC, MBS, etc.|
|Imidate||Lys to Lys||suberimidate, adipimidate|
|Table II. List of common carrier proteins.|
|Keyhole limpet hemocyanin (KLH)||Quite antigenic/Background considerations in invertebrates|
|Thyroglobulin||Extremely antigenic/Background considerations in vertebrates|
|Serum Albumin (BSA, RSA, HSA)||Reduced background when albumin source matched to animal|
|Botilinus toxin||Extremely antigenic/Toxic|
|Succinylated proteins||Quite antigenic/Ideal for carbodiimide coupling|
|Polylysine||High coupling efficiency|
This method involves the use of water-soluble carbodiimides to activate carboxyl groups on peptides and proteins (Asp and Glu residues). The activated carboxyl groups then react with amino groups (on Lys or the amino terminus) to form a stable amide bond. This method is very effective but can be complicated by the presence of both amino and carboxyl groups in most peptides and proteins. The polymerization which can result is a complication which can be partially addressed by selecting peptides with single amino or carboxyl groups or the use of protecting groups when feasible (citraconylation of amino groups, acetylation of the amino terminus, or amidation of the C-terminus). The use of succinylated carrier proteins also minimizes polymerization. Despite the problems with this method, it is quite straightforward and usually leads to a high degree of derivatization. Even when excessive polymerization occurs, the conjugate is usually still a good antigen.
1. Mix 10 mg of carrier with 1-5 mg of peptide antigen in 10 ml of distilled water. Make certain that both components are completely dissolved.
2. Add 100 mg of water soluble carbodiimide (e.g. , EDAC). Incubate 3 hours to overnight at RT. You will usually see a precipitate form. Do not discard this. Your antigen/carrier conjugate will probably be distributed between a soluble and an insoluble form. Both are suitable for antibody production. Note that you should check the pH (use pH paper) occasionally to verify that it has not dropped below 4. If it has, then adjust it with small amounts of base (NaOH or KOH). The ideal pH is 5-6.
3. Dialyze 24 hours against water or PBS (3-4 changes). Remember to save the precipitate. Freeze at -80 or freeze-dry for storage.
1.) Carbodiimides will form stable adducts with proteins in a side reaction to the normal activation process. The resultant moieties are quite antigenic and are specific for the type of carbodiimide used for formation of the conjugate. For this reason, it is not a good idea to screen serum using a conjugate formed using the same water soluble carbodiimide. For example, if you employ EDAC to form the conjugate for inoculation, use CHMC to form the conjugate for screening.
2.) The carbodiimide method is a high success rate method in terms of forming a conjugate successfully. Just make certain that your carbodiimide is still good. It does decompose with time in the presence of moisture.
3.) You can quench the reaction by addition of sodium acetate, sodium propionate, etc. if desired, but it is not necessary.
Bis-diazotized benzidine (diazo) Coupling for His, Tyr, and Lys residues.
Step 1, Activation.
Incubate 100 mg of benzidine and 76 mg sodium nitrite (NaNO2) in 21.8 ml of 0.2 N HCl for 60 minutes at 4 degrees. Store up to one week by storage at -20 degrees.
Step 2, Coupling reaction.
1. Dissolve 20 mg of carrier protein and 5 mg of antigenic peptide in 1.5 ml of .13 M sodium chloride, 0.16 M sodium borate, pH 9.0. Make certain that the peptide and protein are completely dissolved.
2. Add 1 ml of bis-diazotized benzidine.
3. Incubate at 4 degrees for 2-4 hours. Formation of a dark, reddish-brown precipitate is typical.
4. Dialyze against water for 24 hours with 3 changes.
6. Freeze dry or freeze solution for long-term storage.
1.) Cross-linking specificity towards His is improved at pH 8.0 (Lys is protonated, but His is not at this pH).
2.) Lys residues can be temporarily protected if necessary by citraconylation.
3.) The reaction can be quenched by addition of tyrosineamide or acetyl-tyrosine, but this is not normally necessary.
Glutaraldehyde is a homobifunctional crosslinker which reacts with amino groups of lysyl residues and the amino termini of both the antigenic peptide and the carrier. For this reason, the same crosslinking problems observed using other homobifunctional reagents or carbodiimides can occur. The method is fairly reliable and should be particularly considered when glutaraldehyde is used as a tissue fixative.
1. Dissolve 10 mg of carrier protein and 5 mg of peptide antigen in 10 ml of PBS.
2. Add 10 microliters of glutaraldehyde with mixing.
3. Incubate 2 hours at room temperature.
4. Terminate by addition of 50 microliters of 1 M Tris, pH 8.
5. Dialyze the reaction mixture against PBS or water for 24 hours with three to four changes of buffer.
1.) Precipitation is not an uncommon occurrence with this method. It is not usually a problem in antibody production.
Heterobifunctional crosslinkers have the advantage of providing greater control over the crosslinking than methods which rely on homobifunctional crosslinkers. Two frequently crosslinkers, MBS and SMCC , contain an activated carboxyl group at one end which can react with amino groups and a maleimido group at the opposite end which reacts readily with the sulfhydryl group of cysteine residues. Typically, peptide antigens are synthesized with a single cysteine residue at either the N- or C-termini. It< is best to avoid peptides containing internal Cys residues. The crosslinker is first reacted with the amino groups on the carrier protein, followed by removal of the unreacted crosslinker using a desalting column or dialysis. The activated carrier protein is then reacted with the Cys-containing peptide antigen.
Step 1, Preparation of the Activated Carrier Protein.
1. Dissolve 10 mg of the carrier protein in PBS.
2. Add 2 mg of MBS or SMCC dissolved in 200 microliters of DMF.
3. Incubate at room temperature for 2 hours with stirring.
4. Dialyze against a large volume of water overnight.
5. Store frozen at -20 for up to one month.
Step 2, Coupling of the Peptide Antigen to the Activated Carrier.
1. Dissolve 10 mg of peptide in 1-2 ml of PBS. If peptide is only partially soluble, increase the volume
to no more than 3 ml. Solubility may be enhanced by addition of up to 50% DMF or DMSO. Do not
leave the peptide in DMSO for very long to avoid oxidation.
2. Add 10 mg of activated carrier protein to the peptide solution.
3. Incubate four hours at room temperature.
4. Dialyze against water overnight and then freeze-dry for storage.
1.) Freshly synthesized peptide antigen frequently has very little of the oxidized form of the peptide (disulfide linked dimer), and can be used directly. Only the reduced form of the peptide will react with the maleimido groups on the activated carrier. The oxidation occurs at a greatly increased rate in solution. If your peptide antigen is old or has been stored in solution, you must verify the presence of sulfhydryl groups using Ellmans reagent or include a reduction step in your protocol. Inclusion of 0.2% tributylphosphine in the coupling reaction is usually sufficient. Remember that tributylphosphine is quite toxic and must be used in a chemical hood. Alternatively, the peptide antigen may be reduced using dithiothreitol and then thoroughly desalted before use. You must remove all traces of DTT prior to coupling.
2.) The stoichiometry of coupling is difficult to determine for these complexes. One method is to incorporate one beta-alanine residue immediately adjacent to the cysteine residue on the peptide antigen. Amino acid analysis may then be used to determine the amount of beta-alanine present after dialysis of the conjugate.
Succinylation of Carrier Proteins:
Succinylation is a permanent way to replace all amino groups on a protein by carboxyl groups. Succinylated carrier proteins provide a number of advantages for antibody production over underivatized proteins: Increased number of carboxyl groups for activation, resulting in an increased level of antigen coupling. Acylation of amino groups decreases the amount of polymerization during carbodiimide coupling. Changes in the normal epitopes of the carrier protein may reduce the amount of background reactivity of the antiserum.
1. Dissolve 300 mg protein in 100 ml phosphate-buffered saline (PBS) in 250 ml beaker and stir gently.
2. Monitor pH throughout the reaction and maintain at pH 8.0 by dropwise addition of 1.0 N NaOH.
3. Add 1.2 grams of succinic anhydride solid slowly over 30 minutes. The pH will begin to drop immediately.
4. Allow solution to stir gently at room temperature for two hours after pH has stabilized.
5. Dialyze exhaustively against water and freeze dry for storage.
The succinylation reaction may be run in 6 M urea to increase coupling yields, but some proteins may not be soluble after dialysis.
1.) MBS-Kitagawa, T. and Aikawa, T. (1976) J. Biochem. 79, 233.
2.) SMCC-Yoshitake, S., et al. (1979) Eur. J. Biochem. 101, 395-399.
3.) Carbodiimide-Bauminger, S. and Wilchek, M. (1980) in Methods in Enzymology 70, 151-159.
5.) Glutaraldehyde-Reichlin, M. (1980) in Methods in Enzymology 70, 159-165.
6.) Andrews, Picomethods
Tributylphosphine (toxic and pyrophoric as the neat liquid when in contact with cellulose).
Glutaraldehyde (toxic, must be used in a hood).
Carbodiimides will cause chemical burns. It is also a common occurrence for investigators to become highly sensitized to them. Use gloves and other appropriate precautions.
Benzidine is considered a cancer suspect agent. Consider using the prepackaged 100 mg vials of benzidine from Sigma Chemical Co.
The following are some references for conjugation and antibody production:
Bioconjugate Techniques, 1996, by G.T.Hermanson Acad.Press.
Chemistry of Protein Conjugation and Cross-linking, S.S.Wong, 1991 CRC Press
Chemical Reagents for Protein Modification, R.L.Lundblad, 2nd Ed. 1991
Nonradioactive Labeling and Detection of Biomolecules, C.Kessler, Springer Verlag 1992.
Chemical Modification of Proteins, G.E.Means and R.E.Feeney Holden-Day, 1964
Bioconjugate Chemistry, ACS (periodical)
The Pierce Chemical Co. catalog has a lot of good information.
"Photochemical conjugation of peptides to carrier proteins using 1,2,3 thiadiazole-4-carboxilic acid" P.R. Hansen et al. Int J. Pep and Prot Research, vol. 47 no. 6 June 1996.
BioTechniques, Vol. 25, No. 1, July 1998 "Direct Use of synthetic peptides for antiserum production" (via incorporation of a 6-his tag into a peptide sequence)