Expression and purification methods for the production of recombinant human complement component C2.

Human complement component C2 is a critical factor of the classical complement pathway. Here we provide a method for the production of recombinant human C2 (rhC2) protein for research purposes. The human complement component C2 (hC2) is cloned from a human cDNA library by polymerase chain reaction and inserted in a mammalian expression vector (Martini et al., BMC Immunol 11:43, 2010). Transient transfection is utilized to express hC2 in a mammalian cell line, and the expressed C2 is harvested from the conditioned media. rhC2 is purified from the conditioned media by sequential steps of cation exchange and affinity column chromatography. The purified hC2 is characterized for protein purity, stability, and enzymatic activity. The recombinant hC2 activity is tested in a complement activation ELISA assay that measures classical, alternative, and lectin complement pathway activity in C2-depleted serum.

Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases.

BACKGROUND: Complement C2 deficiency is the most common genetically determined complete complement deficiency and is associated with a number of diseases. Most prominent are the associations with recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults. The links with these diseases reflect the important role complement C2 plays in both innate immunity and immune tolerance. Infusions with normal fresh frozen plasma for the treatment of associated disease have demonstrated therapeutic effects but so far protein replacement therapy has not been evaluated. RESULTS: Human complement C2 was cloned and expressed in a mammalian cell line. The purity of recombinant human C2 (rhC2) was greater than 95% and it was characterized for stability and activity. It was sensitive to C1s cleavage and restored classical complement pathway activity in C2-deficient serum both in a complement activation ELISA and a hemolytic assay. Furthermore, rhC2 could increase C3 fragment deposition on the human pathogen Streptococcus pneumoniae in C2-deficient serum to levels equal to those with normal serum. CONCLUSIONS: Taken together these data suggest that recombinant human C2 can restore classical complement pathway activity and may serve as a potential therapeutic for recurring bacterial infections or SLE in C2-deficient patients.