Recombinant Bison bonasus Pregnancy-associated glycoprotein 71D

What is Bison bonasus Pregnancy-associated glycoprotein 71D and how does it compare to bovine PAGs?

Bison bonasus Pregnancy-associated glycoprotein 71D (BbPAG-71D) is a member of the aspartic proteinase family secreted by trophoblast cells of the European bison placenta during pregnancy. Similar to bovine PAGs, BbPAG-71D likely appears in maternal circulation when giant binucleate trophoblast cells (BNCs) migrate across the microvillar junction to fuse with uterine epithelium around days 19-21 of gestation .

While specific data on BbPAG-71D is limited, we can infer from bovine research that these glycoproteins likely play essential roles during pregnancy and serve as potential biochemical markers of pregnancy in European bison. Given the phylogenetic relationship between Bison bonasus and Bos taurus (both within the genus Bos according to modern genetics ), these proteins likely share significant sequence homology, particularly in catalytic domains and structural motifs.

The molecular weight of the glycosylated form would be expected to be approximately 60 kDa, similar to bovine PAG1, which has been identified in placental extracts .

What expression systems are suitable for producing recombinant BbPAG-71D?

Based on research with bovine PAGs, several expression systems could be employed for BbPAG-71D:

1. Mammalian Cell Expression Systems:

• Human Embryonic Kidney 293 (HEK 293) cells have demonstrated superior expression of glycosylated bovine PAG1 compared to other cell lines .

• These cells can be adapted to suspension culture in spinner flasks for scaled-up production .

• This system would produce fully glycosylated forms that more closely resemble native BbPAG-71D.

2. Bacterial Expression Systems:

• Direct expression of mature PAG in E. coli has proven challenging with bovine PAG1 .

• A fusion protein approach using E. coli thioredoxin (Trx) as a partner significantly improves expression .

• The resulting Trx-PAG fusion protein accumulates in the insoluble fraction and requires appropriate solubilization and refolding .

• This approach produces non-glycosylated protein that may be sufficient for some applications (e.g., generating antibodies).

What methodologies are available for detecting BbPAG-71D in research contexts?

Several methodological approaches can be employed to detect BbPAG-71D in research samples:

1. Enzyme-Linked Immunosorbent Assay (ELISA):

• In-house developed ELISAs using specific antibodies against BbPAG-71D would be the primary detection method.

• Research in cattle demonstrates that ELISA can successfully detect PAG concentrations as early as day 24 post-breeding .

• Threshold values determined through ROC curve analysis (e.g., ≥0.33 ng/mL for cows and ≥0.54 ng/mL for heifers in cattle) provide 95% accuracy in determining pregnancy status .

2. Western Blotting:

• Useful for confirming antibody specificity and detecting PAGs in placental tissues.

• Can identify the ~60 kDa glycosylated form in placental extracts .

• Secondary antibody detection systems must be optimized for sensitivity and specificity.

3. Mass Spectrometry:

• For detailed characterization of protein sequence and post-translational modifications.

• Can confirm the identity of protein bands from immunoblots .

• Essential for comparative studies between native and recombinant forms.

What are the optimal purification strategies for recombinant BbPAG-71D?

Purification strategies differ based on the expression system used:

For Mammalian-Expressed Glycosylated BbPAG-71D:

• Ion-exchange chromatography as an initial capture step

• Pepstatin-sepharose affinity chromatography, leveraging the aspartic proteinase nature of PAGs

• Preparative SDS-PAGE for final purification to homogeneity

For Bacterial-Expressed Trx-BbPAG-71D Fusion Protein:

• Solubilization of inclusion bodies containing the fusion protein

• Purification by combination of chromatographic techniques

• Optional: Cleavage of the fusion partner if required for downstream applications

The purification should be monitored using SDS-PAGE and Western blotting with appropriate antibodies. Mass spectrometry can confirm the identity of the purified protein, as demonstrated with bovine PAG1 .

How can researchers develop specific monoclonal antibodies against BbPAG-71D?

Development of specific monoclonal antibodies (mAbs) against BbPAG-71D requires a strategic approach:

1. Antigen Preparation:

• Express recombinant BbPAG-71D using the thioredoxin fusion approach in E. coli .

• Purify to high homogeneity for immunization.

2. Hybridoma Production and Screening:

• Immunize mice with the purified recombinant protein.

• Screen hybridoma supernatants against the recombinant protein.

• Counterscreen against the fusion partner (Trx) to eliminate antibodies targeting it rather than BbPAG-71D.

3. Validation Studies:

• Test antibody reactivity with placental extracts via Western blotting.

• Confirm specificity through mass spectrometry identification of the recognized protein .

• Evaluate cross-reactivity with related PAGs from other species.

4. Application Development:

• Optimize antibody pairs for sandwich ELISA development.

• Validate the assay using serum samples from confirmed pregnant European bison.

Research with bovine PAG1 has demonstrated that this approach can successfully yield mAbs that recognize both recombinant protein and the native protein in placental extracts .

What challenges exist in expressing glycosylated forms of BbPAG-71D?

Producing properly glycosylated recombinant BbPAG-71D presents several significant challenges:

1. Expression System Limitations:

• Mammalian cell systems (HEK 293, CHO) are required for native-like glycosylation .

• These systems typically yield lower protein amounts compared to bacterial systems.

• Bacterial systems (E. coli) cannot perform glycosylation, producing proteins that may differ structurally and functionally from native forms .

2. Glycosylation Heterogeneity:

• Even in mammalian systems, glycosylation patterns may differ from native BbPAG-71D.

• Batch-to-batch variation can occur, requiring consistent monitoring.

3. Scale-Up Challenges:

• Transitioning from adherent to suspension culture requires optimization to maintain protein quality .

• Maintaining consistent glycosylation during scale-up is technically challenging.

Research with bovine PAG1 has shown that HEK 293 cells are superior to CHO cells for expressing glycosylated forms , suggesting this would be the preferred system for BbPAG-71D expression when glycosylation is important.

How do PAG concentrations differ throughout pregnancy in Bison bonasus compared to domestic cattle?

While specific data on PAG concentration profiles in European bison might be limited, bovine research provides a framework for investigation:

Temporal Profile in Cattle:

• PAGs can be detected as early as day 24 of gestation in cattle .

• Pregnant heifers show significantly higher PAG concentrations (3.29 ± 0.36 ng/mL) compared to pregnant cows (1.39 ± 0.10 ng/mL) at day 24 .

• The probability of pregnancy increases as circulating PAG concentration increases .

Factors Affecting PAG Concentrations:

• Parity (first pregnancy vs. subsequent pregnancies)

• Age of the animal

• Embryonic mortality can be associated with decreased PAG concentrations

Research Approach for Bison bonasus:

• Establish sampling protocols for European bison in conservation settings

• Validate PAG assays specifically for Bison bonasus

• Conduct longitudinal studies to map concentration changes throughout pregnancy

• Compare patterns with established bovine PAG profiles

This type of comparative research would significantly enhance understanding of reproductive physiology in European bison and improve pregnancy monitoring in conservation programs.

How can recombinant BbPAG-71D contribute to conservation efforts for European bison?

Recombinant BbPAG-71D could significantly advance European bison conservation through several applications:

1. Non-Invasive Pregnancy Monitoring:

• Development of specific BbPAG-71D assays would enable blood-based pregnancy detection.

• This would minimize handling stress compared to physical or ultrasound examination.

• Early detection (potentially by day 24 as in cattle) would improve reproductive management in conservation breeding programs .

2. Reproductive Health Assessment:

• Changes in PAG profiles could potentially indicate embryonic mortality or pregnancy complications.

• In cattle, animals experiencing late embryonic mortality show decreased PAG concentrations .

• Similar patterns could be monitored in European bison to identify at-risk pregnancies.

3. Research Applications:

• Immunological reagents developed against recombinant BbPAG-71D would enable comparative studies with other bovid species.

• Enhanced understanding of reproductive biology in this threatened species.

• Development of species-specific pregnancy tests tailored to conservation needs.

The availability of recombinant BbPAG-71D would be "useful for creating immunological reagents to detect pregnancy in [European bison], which is of great practical importance" for conservation management.