ABSTRACT
Recombinant human zona pellucida (rhZP) proteins (minus the N-terminal leader and the C-terminal transmembrane-like domain) were expressed in four different expression systems: bacteria, yeast, insect cells, and Chinese Hamster Ovary (CHO) cells. The recombinant proteins in each system were engineered with a C-terminal six histidine (His6) segment that was used to purify the proteins by metal affinity [either nickel (Ni) or cobalt (Co)] column chromatography. Each of the rhZP proteins was a candidate antigen as an immunocontraceptive vaccine. However, the rhZP proteins produced in bacteria, yeast and insect cell culture could only be purified after being solubilized by strong denaturants. After purification the final products of each of these expression systems required 6 M urea to maintain solubility. However, the rhZP proteins expressed by CHO cells were secreted into the media, and the soluble proteins could be purified to near homogeneity. In this report the expression and purification procedures used to produce and isolate these secreted proteins are described.
Subject(s)
Egg Proteins/genetics , Membrane Glycoproteins/genetics , Receptors, Cell Surface , Animals , Blotting, Western , CHO Cells , Chromatography, Liquid , Cricetinae , Egg Proteins/isolation & purification , Electrophoresis, Polyacrylamide Gel , Humans , Membrane Glycoproteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae/genetics , Zona Pellucida GlycoproteinsABSTRACT
The zona pellucida (ZP) is an extracellular matrix composed of multiple glycoproteins that surrounds all mammalian oocytes. Analysis of the genes encoding the ZP proteins indicates that there are three classes ZPA (largest), ZPB (intermediate) and ZPC (smallest). Polyclonal antibodies developed against recombinant proteins of human and pig ZP have been used to identify immunohistochemically the components of the ZP of cynomolgus monkeys during the course of follicular development. Digoxigenin-labelled cDNA probes specific for the mRNA encoding ZPA, ZPB or ZPC were used to probe sections of cynomolgus monkey ovaries by in situ hybridization. mRNA encoding ZPA was identified in growing follicles at all stages and was seen in the granulosa cells of mature preovulatory follicles. ZPB protein and mRNA encoding ZPB were present in oocytes in secondary follicles and to a lesser extent in tertiary follicles, but were not found in primordial, primary or antral follicles or granulosa cells. ZPC protein and mRNA encoding ZPC were present in oocytes at all stages of folliculogenesis. These results suggest that each component of the ZP is produced and secreted at specific stages of folliculogenesis and that granulosa cells may be contributing to the synthesis of ZPA and ZPC.
Subject(s)
Egg Proteins/genetics , Membrane Glycoproteins/genetics , RNA, Messenger/analysis , Receptors, Cell Surface/genetics , Zona Pellucida/metabolism , Animals , Egg Proteins/isolation & purification , Female , Gene Expression , Immunohistochemistry , In Situ Hybridization , Macaca fascicularis , Membrane Glycoproteins/isolation & purification , Receptors, Cell Surface/isolation & purification , Zona Pellucida GlycoproteinsABSTRACT
Full length zona pellucida cDNAs from cat, dog and pig that are homologous to the ZP2/rc75 genes from mouse, human and rabbit, a full length zona pellucida cDNA from cat and a gene and full length cDNA from human that are homologous to the rc55/ZP3 alpha genes from rabbit and pig, and full length zona pellucida cDNAs from cat, cow, dog, pig and rabbit that are homologous to the ZP3 genes from mouse, hamster, human and marmoset have been cloned and characterized. The members of these gene families are herein referred to as ZPA, ZPB and ZPC genes to avoid the confusion that currently exists in the zona pellucida of nomenclature. This report is the first to describe the presence all three major zona pellucida genes within individual mammalian species. Within the ZPA, ZPB and ZPC gene families, the DNA and deduced amino acid sequences are highly homologous to each other, and are most homologous between members of the same order within the class mammalia. These results imply that all or most mammalian species express the ZPA, ZPB and ZPC proteins, which form the zona pellucida layer surrounding the oocyte.
Subject(s)
Egg Proteins/genetics , Membrane Glycoproteins/genetics , Receptors, Cell Surface , Zona Pellucida , Animals , Base Sequence , Cats , Cattle , Cloning, Molecular , Cricetinae , DNA, Complementary , Dogs , Humans , Mammals , Mice , Molecular Sequence Data , Oocytes , Rabbits , Sequence Homology , Zona Pellucida GlycoproteinsABSTRACT
We isolated a cDNA encoding the pig oocyte zona pellucida protein ZP3 alpha from a pig ovary lambda gt11 cDNA library. The 1699 bp cDNA contains a short 3' untranslated region characteristic of cDNAs encoding zona proteins. The deduced amino acid sequence for ZP3 alpha consists of 536 amino acid residues and shares 66% overall identity with a 55 kDa rabbit zona protein. Important features of the ZP3 alpha polypeptide include a predicted N-terminal signal sequence, twenty-two cysteine residues, an O-glycosylated domain and potential attachment sites for five N-linked sugar chains. A multibasic tetrapeptide occurs upstream of a predicted C-terminal transmembrane sequence; this suggests proteolytic processing of an integral membrane precursor within the constitutive secretory pathway.
