| Autor: |
Norton AW; Department of Biomolecular Research and Technologies, Shire Human Genetic Therapies, Cambridge, MA, USA., Martini PG, Cook LC, Alderucci S, Lundberg DM, Fish SM, Bedard C, Gill J, Tzianabos AO, Concino MF |
| Jazyk: |
angličtina |
| Zdroj: |
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2014; Vol. 1100, pp. 61-74. |
| DOI: |
10.1007/978-1-62703-724-2_5 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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