Production and Purification of Recombinant Human Oviduct-Specific Glycoprotein (rhOGP) and the Role of rhOGP in Sperm Capacitation and Acrosome Reaction.
Bibliographic record
Abstract
The secretory cells lining the lumen of mammalian oviducts synthesize and secrete a major glycoprotein known as oviduct-specific glycoprotein (OGP) or oviductin which has been shown to interact with gametes and early embryos. We report here the use of recombinant DNA technology to produce human OGP in HEK-293 cells. A full-length cDNA clone of human OGP was constructed in an expression vector pCMV6. For the establishment of stable colonies expressing human oviductin, HEK-293 cells were transfected with human OGP cDNA. Single colonies were obtained by limiting dilution in medium supplemented with Genecticin. Colonies were screened for expression levels by immunoblot analysis of culture supernatants. HEK-293 cells stably expressing recombinant human OGP (rhOGP) were maintained in the medium supplemented with Genecticin. We utilized the CELLSPIN cell cultivation system to produce large quantities of rhOGP in stably transfected cells. Production of rhOGP from the culture medium was monitored by immunoblotting. The medium was collected and concentrated by dialysis against polyethylene glycol. The concentrated medium was further dialyzed against phosphate buffer prior to initiating cation exchange chromatography. Fractions containing the eluted proteins were analyzed by immunoblotting. These fractions were then pooled and concentrated by centrifugal filtration for size exclusion chromatography. Fractions were collected in the size range of oviductin. Upon gel electrophoresis, purified rhOGP yielded a single band corresponding to the 120-150 kDa size range of human OGP reported in literature. Subsequent mass spectrometric analysis of the purified rhOGP revealed its identity as human oviduct-specific glycoprotein. Preliminary in vitro functional studies showed that addition of rhOGP in the capacitation medium further enhance tyrosine phosphorylation of human sperm proteins in a time-dependent manner. After 4 hours of incubation in the presence of rhOGP, an increase in both spontaneous and A23187-induced acrosome reaction was measured. The successful production of rhOGP can now facilitate the study of the role of human OGP in fertilization and early embryo development. (Supported by the Canadian Institutes of Health Research - Grant #MOP93606) (poster)
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".