Overexpression, purification, and characterization of third component of complement

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• The third component of complement (C3) is crucial for both classical and alternative complement pathways.
• Understanding C3's molecular features is key to elucidating its functional activities.

Purpose of the Study:

• To express and characterize recombinant human C3 (rC3) using a baculovirus expression system.
• To assess the functional activity and structural integrity of the expressed rC3.

Main Methods:

• Human C3 cDNA was expressed in insect cells via a baculovirus vector (AcNPV).
• Recombinant C3 (rC3) was purified and analyzed using SDS-PAGE and Western blotting.
• Glycosylation patterns were assessed, and reactivity with monoclonal antibodies and complement-binding proteins was evaluated.
• Functional assays included reconstitution of C3-deficient serum and support of complement pathway activation.

Main Results:

• Baculovirus-mediated expression yielded approximately 10 µg/ml of rC3.
• rC3 consisted of 115 kDa and 73 kDa chains, similar to serum-derived C3 (sC3), and exhibited analogous glycosylation.
• rC3 demonstrated conformational similarity to native C3 and reacted with C3-binding proteins (CR1, Factor P, Factor H).
• Purified rC3 reconstituted C3-deficient serum and supported both classical and alternative complement pathway activation.
• Both C3 and proC3 were found to be susceptible to cleavage by Factor I.

Conclusions:

• The baculovirus expression system successfully produced functional rC3 with native-like conformation and activity.
• This rC3 can participate in complement activation and is processed by key regulatory proteins (Factor I and H).
• The findings validate the use of this rC3 for further studies on complement system mechanisms and C3 function.