Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line.
OBJECTIVE
To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests.


METHODS
The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed.


RESULTS
RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZP1 was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZP1 was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed.


CONCLUSIONS
RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZP1 from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.
Martic
Mirjana
M
Department of Obstetrics and Gynaecology, University of Melbourne, 320 Victoria Parade, East Melbourne 3002 VIC, Australia. marticm@iprimus.com.au
Moses
Eric K
EK
Adams
Tim E
TE
Liu
De Yi
DY
Gook
Debra A
DA
Garrett
Claire
C
Dunlop
Marjorie E
ME
Baker
Gordon H W
GH
D020101:Acrosome Reaction / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D004527:Egg Proteins / Q000032:analysis / Q000235:genetics* / Q000494:pharmacology; D004622:Embryo, Mammalian; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics* / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000494:pharmacology; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606514:ZP1 protein, human; C000606515:ZP2 protein, human; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; D006026:Glycoside Hydrolases