Toward a Cell-Based Assay for Cubam-Mediated Intrinsic Factor-Vitamin B12 Binding and Uptake

Background: Vitamin B12 deficiency caused by intrinsic factor (IF) autoantibodies can lead to pernicious anemia. The 2025 iGEM project aimed to develop a novel oral B12 supplement capable of escaping autoantibody recognition. This study was designed to complete and continue the iGEM project with the ultimate goal to establish a Ba/F3 cell model for evaluating IF-Cubam receptor interactions predictive of functionality in the human digestive tract. Methods: A Cubam/EpoR chimeric receptor was expressed on the cell surface, which was expected to activate the JAK-STAT5 pathway and drive cell proliferation upon binding to the IF-Cbl complex. The main tasks included cloning and expressing human, bovine, and platypus IF in Komagataella phaffii (previously known as Pichia pastoris) strain X-33; constructing the AMN expression plasmid and the mini Cubilin-EGFP fusion plasmid; and co-transfecting both plasmids into Ba/F3 cells to complete the model construction. Results: Yeast expression vectors and stable transformants for human, bovine, and platypus IF were successfully obtained, but no conclusive evidence of secreted IF expression was obtained by SDS-PAGE, although very faint bands were observed in the expected molecular weight range. Possible explanations include signal peptide processing defects, limited secretory capacity, or degradation. The AMN plasmid was successfully constructed. The mini Cubilin-EGFP plasmid was successfully constructed using a two-step cloning strategy, and whole-plasmid sequencing confirmed the correct reading frame, with only one synonymous mutation (c.1067C>T) that did not affect the amino acid sequence. Conclusions: This study successfully constructed the two key plasmids required for the Ba/F3 cell Cubam/EpoR chimeric receptor model, laying the molecular foundation for subsequent co-transfection and functional validation. However, due to the failure to replicate previously reported rat and bovine IF expression and to express human and platypus IF in K. phaffii, ligand-dependent functional assays could not yet be performed. Future work will optimize IF expression; once functional IF is obtained, co-transfection into Ba/F3 cells can be carried out to validate the signaling capability.

Grantor dc:publisher

Helsingin yliopisto

Year dc:date.issued

2026

Author dc:creator

• Gong, Cao

• Vitamin B 12 Deficiency
• Intrinsic Factor
• Cubilin
• Amnionless
• Chimeric Receptor

Statement dc:rights

• In Copyright 1.0

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/633409

OAI identifier oai:identifier

oai:helda.helsinki.fi:10138/633409