Purification and characterization of human cell--cell adhesion molecule 1 (C-CAM1) expressed in insect cells

Area of Science:

• Molecular Biology
• Oncology
• Immunology

Background:

• Cell-cell adhesion molecule 1 (C-CAM1) functions as a tumor suppressor, particularly in prostate cancer.
• Reduced C-CAM1 expression is observed in various carcinomas, including prostate, breast, and colon.
• Re-expression of C-CAM1 inhibits tumor growth in preclinical models, suggesting its potential as a diagnostic marker.

Purpose of the Study:

• To produce a sufficient quantity of purified human C-CAM1 for monoclonal antibody generation and biochemical analysis.
• To validate the functional and biochemical characteristics of the expressed C-CAM1 protein.

Main Methods:

• Overexpression of full-length human C-CAM1 in Sf9 insect cells using a baculovirus expression system.
• Purification of C-CAM1 using nickel affinity chromatography.
• Characterization of the purified protein through SDS-PAGE, deglycosylation assays, Western blotting, and protein sequencing.

Main Results:

• Purified C-CAM1 protein was obtained with 104-fold enrichment and a yield of 0.4 mg from 200 mg of infected cells.
• Deglycosylation analysis indicated C-CAM1 is N-linked glycosylated in Sf9 cells, with its molecular weight shifting from 90 kDa to 58 kDa.
• Western blotting and protein sequencing confirmed the identity of the purified protein as human C-CAM1.
• Biochemical and functional assays demonstrated that the expressed C-CAM1 retains adhesion properties and glycosylation modifications similar to the native protein.

Conclusions:

• The baculovirus expression system provides a viable method for producing purified, functional human C-CAM1.
• The produced C-CAM1 protein is suitable for generating specific monoclonal antibodies and conducting further biochemical studies.
• This work facilitates the development of C-CAM1-based cancer detection and therapeutic strategies.