Conjugation Of Synthetic Peptides Research Articles

Thiazolidine Formation as a General and Site-Specific Conjugation Method for Synthetic Peptides and Proteins

A general and mild method for the conjugation of synthetic peptides and proteins through site-specific thiazolidine formation was developed. This method is based on the specific reaction of the 1,2-aminothiol of cysteine with an aldehyde under acidic conditions to form a stable thiazolidine product. The carbonyl precursors are derived from the 1,2-aminoalcohols of Ser or Thr or the vicinalcis-diols of glycoproteins, which can be selectively and rapidly converted to the aldehyde form by periodate oxidation. Since the position where the aldehyde is generated can be manipulated or predetermined, the thiazolidine formation is essentially site-specific. The potential of this method is demonstrated by the biotinylation of three synthetic peptides at the N-terminal Ser or Thr and at the carbohydrate sites of α1-acid glycoprotein. 1-Biotinyl-6-cysteinyl-hexadiamide, a new, water-soluble, long-chained, 1,2-aminothiol derivative containing biotin, was prepared as a capture device. This reagent reacted rapidly with periodate-oxidized N-terminal Ser or Thr peptides and with glycoproteins under very mild conditions to form the thiazolidine conjugates. Based on our results, the thiazolidine conjugation method would be useful for the attachment of radiolabels, reporter groups, imaging agents, and cytotoxic drugs to peptides and proteins.

Conjugation of synthetic peptides to carrier proteins for cell adhesion studies

The ability to reproduce active site sequences from integrin receptor ligands as synthetic peptides has revolutionized the field of cell adhesion. Most studies with these peptides use them in soluble form as inhibitors of integrin function, however, other studies necessitate immobilization of the peptides onto plastic or glass surfaces for analyzing the promotion of cell adhesion. Unfortunately, peptides rarely adsorb well and adsorbed peptides may have their active residues masked by the substrate. An alternative approach is therefore to analyze peptide activity after covalent conjugation to an inert carrier protein. Methods for the production and characterization of peptide conjugates are described.

Conjugation of synthetic peptides to carrier iscoms: factors affecting the immunogenicity of the conjugate

This study was designed to explore optimal conditions for the conjugation of a synthetic peptide to preformed influenza virus iscoms using MHS (maleimidohexanoyl- N-hydroxy-succinimide ester) as coupling agent. The peptide used in this study comprised amino acids 122–138 of porcine growth hormone (pGH). Different ratios of peptide to carrier iscoms were tested and the resulting conjugates were analysed for composition, antigenicity and immunogenicity. The problem of low solubility and poor immunogenicity of high-density peptide conjugates is discussed and a general protocol for conjugation of peptides to carrier iscoms is proposed.

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic peptide conjugates with horseradish peroxidase and β-galactosidase for use in epitope-specific immunocytochemistry and ELISA

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-β-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to β-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1–30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.

An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method

Controlled and efficient conjugation of synthetic peptides to proteins, for use in immunization or in assay procedures, is a prerequisite for the immunological applications of synthetic peptides. This study describes a new method of conjugating synthetic peptides to proteins in such a way that no homopolymers of synthetic peptides or proteins occur. To achieve this, the protein is first activated with glutaraldehyde and subsequently excess glutaraldehyde is removed. Then coupling of the synthetic peptide to the activated protein occurs while subsequetly the surplus reactive glutaraldehyde groups on the protein are blocked with lysine. Excess free peptide and lysine is then removed by dialysis. This improvement not only results in better defined conjugates when compared to classical glutaraldehyde coupling, but also in the consumption of smaller amounts of synthetic peptide during conjugate formation. When used for immunization we obtained similar and sometimes even better responses with the glutaraldehyde based conjugates than with succinimidyl (MBS) conjugates of the same peptides. The performance of the modified conjugates in ELISA procedures, immunization and immunocytochemistry suggests that they are superior to conjugates formed by classical glutaraldehyde coupling.

Conjugation of synthetic peptides to proteins: Quantitation from S-carboxymethylcysteine released upon acid hydrolysis

A method described here for conjugating synthetic peptides to carrier proteins provides a convenient method for determining peptide-to-carrier protein rations. N-Bromoacetyl-containing peptides are reacted in situ with carrier proteins in which the disulfide bonds were reduced with tri- n-butylphosphine. At pH 7–8 and ambient temperature, the newly formed sufhydryl groups of the carrier protein react exclusively with the bromoacetyl moiety of the peptide to form conjugates having stable thio ether linkages. Acid hydrolyses of these conjugates release S-carboxymethylcysteine in amounts proportional to the amounts of peptides conjugated and thus allow determination of peptide-to-protein ratios.

Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus

The predictive algorithm Surfaceplot (J.M.R. Parker, D. Guo, and R.S. Hodges, Biochemistry 25:5425-5432, 1986) was used to examine glycoprotein D of herpes simplex virus type 1 (HSV-1) for amino acid residues with a high probability of being exposed on the molecular surface. Based on these data, 11 different peptides corresponding to 10-residue segments in the primary sequence of glycoprotein D and one 20-residue segment were synthesized, conjugated to carrier proteins, and used to generate specific antisera in rabbits. Two synthetic peptides predicted not to be on the surface of glycoprotein D were included as negative controls. The polyclonal antisera against individual synthetic peptide conjugates were in turn evaluated for their ability to recognize both isolated glycoprotein D and intact HSV-1 virions in an enzyme-linked immunosorbent assay. Based on Surfaceplot predictions, eight linear antigenic sites on glycoprotein D were thereby defined from the 12 antipeptide antisera prepared. Four of these sites contained epitopes to which complement-independent neutralizing antibodies could be generated. The latter sites corresponded to sequences 12 to 21, 267 to 276, 288 to 297, and 314 to 323 of the mature protein. An additional peptide sequence, 2 to 21, was found to generate antisera which had potent virus-neutralizing capacity in the presence of complement. Identification of a neutralizing epitope in the sequence 314 to 323 makes it likely that the membrane-spanning region of glycoprotein D is within the subsequent sequence, 323 to 339. Antipeptide antisera prepared in this study from 12 synthetic peptides contained 13 surface sites predicted by Surfaceplot, of which 7 were not predicted by the parameters of Hopp and Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981). Of these seven sites not predicted by the Hopp and Woods plot, all generated antipeptide antibodies that bound to HSV-1 virions and three of these seven sites generated neutralizing antibodies. In total, 8 of 12 synthetic peptides containing surface regions produced antipeptide antibodies that bound to HSV-1 virions and 5 of these generated neutralizing antibodies. These results suggest the advantages of Surfaceplot in mapping antigenic determinants in proteins.

Antigen-antibody interaction. The immunodominant region of EDP208 pili.

The EDP208 pilus contains a major antigenic determinant in the N-terminal dodecapeptide, as shown by E. A. Worobec, A. K. Taneja, R. S. Hodges, and W. Paranchych ((1983) J. Bacteriol. 153, 955-961). This peptide was chemically synthesized, coupled to bovine serum albumin with N-hydroxysuccinimidyl p-azido-benzoate, and used in immunoblot and enzyme-linked immunosorbent assays to show it was capable of reacting with anti-EDP208 pilus antibodies. Antibodies raised against the synthetic peptide conjugate were also capable of reacting with whole pili in these assays. To further examine the specific residues responsible for the antigenicity of this site, several peptide analogs were chemically synthesized. The relative affinity of these peptides for anti-EDP208 pilus antibodies was determined by a competitive enzyme-linked immunosorbent assay using the Fab fragment of anti-EDP208 pilus immunoglobulin G. From these results we established that the antigenic region of this peptide was the N-terminal pentapeptide, N-acetyl-Thr-Asp-Leu-Leu-Ala, and the key residues responsible for the antibody-antigen interaction are the N-acetyl-Thr1, Leu3, and Leu4. Hydrophobic interactions involving the methyl of the acetyl group and the leucine side chains make the largest contributions to the antigen-antibody interaction, while a lesser contribution is made by the Thr1 hydroxyl. The side chains of Asp2 and Ala5 contribute only weakly to the stabilization of the antigen-antibody complex.