Synthesis and application of peptide immunogens related to the sperm tail protein tpx-1, a member of the CRISP superfamily of proteins.
The synthesis of peptides containing 0, 1 and 2 cysteine residues related to the human sperm tail protein, tpx-1, is described. These synthetic peptides, following conjugation to keyhole limpet hemocyanin modified with maleimidobenzoic acid N-hydroxysuccinimide ester, were used as immunogens to generate polyclonal antibodies in female New Zealand white rabbits. The binding characteristics of the derived antipeptide sera were evaluated using indirect and competitive ELISA procedures. Western immunoblot experiments also confirmed that these synthetic peptide immunogens are able to generate high-titer polyclonal antibodies capable of cross-reacting with the mature tpx-1 protein present in crude rat sperm tail/testis preparations as well as in outer dense fiber preparations. Consequently, these synthetic peptides represent promising candidates for investigations into the role of tpx-1 in the immunoregulation of sperm function in the rat and other mammalian models, with the derived antisera also providing an avenue to explore possible sites of expression of tpx-1 proteins in other tissues.
Keah
H H
HH
Center for Bioprocess Technology, Department of Biochemistry and Molecular Biology Monash University, Clayton, Australia.
O'Bryan
M K
MK
de Kretser
D M
DM
Hearn
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000737:chemistry*; D015153:Blotting, Western; D015815:Cell Adhesion Molecules; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D008297:Male; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D012471:Salivary Proteins and Peptides / Q000737:chemistry; D029589:Seminal Plasma Proteins; D013094:Spermatozoa / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D013997:Time Factors; D014269:Trifluoroacetic Acid / Q000737:chemistry
D000941:Antigens; C062790:CRISP2 protein, rat; C489926:CRISP3 protein, human; D015815:Cell Adhesion Molecules; C486320:Crisp3 protein, rat; D004220:Disulfides; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D010455:Peptides; D012471:Salivary Proteins and Peptides; D029589:Seminal Plasma Proteins; D013388:Succinimides; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D014269:Trifluoroacetic Acid; D003545:Cysteine