Method for the use and synthesis of peptides

Details Method for the use and synthesis of peptides Method for the use and synthesis of peptides

1991-09-06

1996-07-23

Warden, Jill

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Synthesis of peptides

530333, 530336, 530337, 530332, A61K 3802

Patent

active

055390846

DESCRIPTION:

BRIEF SUMMARY
This invention relates to the use of synthetic peptides in the separation of complex mixtures of binding entities, and to the synthesis of peptides.
In International Patent Application PCT/AU84/00039 there is disclosed a method for the simultaneous synthesis of large numbers of peptides. This method is based on the solid phase synthesis of peptides onto polyethylene rods or pins as solid supports. In that patent application, it is pointed out that the technique would be useful for systematically determining the continuous antigenic determinants of various antigens by synthesizing all of the overlapping peptides which could be made from the sequence of the antigen and then testing for the ability of the peptide to bind to a binding entity such as an antibody. In effect, there is disclosed a method for a systematic and very rapid determination of the continuous B cell epitopes of an antigen.
Subsequent research using this method has led to fur%her developments which enhance the usefulness of peptides synthesized on inert rods or similar supports. Theses further developments relate to modifications to the earlier invention which extend its use in systematic studies in the field of immunology. Basically the modifications of the earlier invention allow for two different end results: elution of the binding entity from the peptide/solid support complex and harvesting of the peptide from the solid support.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the course of each of two reactions occuring as a result of two alternative points of attachment to a Lys-Pro linker moiety. In FIG. 1(a), the peptide is bonded to the epsilon-amino group of the lysine and the carboxyl of the proline is bonded to the solid phase support; cyclization between the alpha-amino group of the lysine and the ester moiety of proline liberates a peptide bearing an N-(delta-diketopiperazinyl-butyl)amido moiety. In FIG. 1(b), the site of attachment of the peptide and of the solid phase support is reversed; cyclization produces a peptide bearing an N-(omega-hydroxyalkyl)amido moiety.
FIG. 2a illustrates the reaction (ELISA absorbance) between anti-JGMV serum and each of the possible octapeptides corresponding to residues 1-97 of JGMV capsid protein. FIG. 2(b) illustrates the result of binding of the eluant from FIG. 2(a) with the JGMV capsid protein; FIGS. 2(c) and 2(d) illustrate the result of binding of the eluant from FIG. 2(a) with the SMV-N capsid protein and the WMMV-II capsid protein, respectively.
FIG. 3(a) illustrates the reaction (ELISA absorbance) between anti-SMV-N and each of the possible octapeptides corresponding to residues 1-57 of the SMV-N capsid protein. FIG. 3(b) illustrates the result of binding of the eluant from FIG. 3(a) with the JGMV capsid protein; FIGS. 3(c) and 3(d) illustrate the result of binding of the eluant from FIG. 3(a) with the SMV-N capsid protein and the WMMV-II capsid protein, respectively.
FIG. 4a illustrates the reaction (ELISA absorbance) between anti-WMMV-II and each of the possible octapeptides corresponding to residues 1-66 of the WMMV-II capsid protein. FIG. 4(b) illustrates the result of binding of the eluant from FIG. 4(a) with the JGMV capsid protein; FIGS. 4(c) and 4(d) illustrate the result of binding of the eluant from FIG. 4(a) with the SMV-N capsid protein and the WMMV-II capsid protein respectively.
FIGS. 5(a), 5(b) and 5(c) illustrate the result of a T-cell proliferation assay resulting from exposure to each of the possible decapeptides corresponding to residues 1-161 of the MPB70 protein of M. Bovis BCG.
FIGS. 6(a), 6(b), 6(c), 6(d), 6(e), 6(f), 6(g), 6(h), 6(i), 6(j), 6(k) and 6(l) illustrate the results of T-cell proliferation by all possible 9-mers and 12-mers of the MPB70 sequence in the vicinity of residues 20-30, 72-85, and 150-161 of this peptide.
FIGS. 7(a) and 7(b) provide the HPLC tracing and mass spectrum, respectively of the peptide Ac-VQAAIDYING-.beta.-cyclo(KP).
FIGS. 8(a), 8(b) and 8(c) illustrate the ability of antibodies raised against each possible dedapeptide co

REFERENCES:
Merrifield, Angew. Chem. vol. 24 No. 10 (Oct. 1985) 799-810.
Schoofs et al. J. Immun. vol. 140, 611-616 No. 2 (Jan. 1988) 611-616.
Chou et al. JACS vol. 93 Jan. 13 1971 267-268.

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