Recombinant FbpC-C, FbpC-C R248A, and FbpC-C H252A proteins were expressed and purified as in (20 (link)). Briefly, after elution of the Ni column using the Elution buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 500 mM Imidazole), the proteins were exchanged into 20 mM Tris (pH 8.0), 500 mM NaCl, 10 mM Imidazole using a Desalting 26/10 column (GE Healthcare). The His-tag was then removed by incubation with the tobacco etch virus (TEV) protease and 5 mM β-mercaptoethanol overnight at room temperature. The TEV digested proteins were separated from the cleaved His-tag product on an AKTA Pure 25L FPLC using a 5 mL HisTrap-FF with the captured flowthrough further purified using a HiLoad Superdex 75 PG gel filtration column (GE Healthcare). A monodisperse peak was obtained and assessed for purity using SDS-PAGE gel analysis, then pooled, concentrated, and exchanged into HBS buffer (10 mM HEPES [pH 7.3], 140 mM NaCl). Purified full length zymogen and active forms of C1r were obtained from Complement Technology, Inc. (Tyler, TX).
Active forms of c1r
Manufactured by Complement Technology
Active forms of C1r are components of the complement system, a group of proteins that play a crucial role in the body's immune response. C1r is an enzyme that activates the classical complement pathway, which is involved in the recognition and elimination of pathogens and damaged cells.
Automatically generated - may contain errors
2 protocols using active forms of c1r
1
Purification of FbpC-C Proteins
2
Purification and Characterization of FbpC-C Proteins
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Recombinant FbpC-C, FbpC-C R248A, and FbpC-C H252A proteins were expressed and purified as in (20 ). Briefly, after elution of the Ni column using the elution buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 500 mM imidazole), the proteins were exchanged into 20 mM Tris (pH 8.0), 500 mM NaCl, 10 mM Imidazole using a Desalting 26/10 column (GE Healthcare). The His-tag was then removed by incubation with the tobacco etch virus protease and 5 mM β-mercaptoethanol overnight at room temperature. The tobacco etch virus–digested proteins were separated from the cleaved His-tag product on an AKTA Pure 25L FPLC using a 5 ml HisTrap-FF with the captured flowthrough further purified using a HiLoad Superdex 75 PG gel filtration column (GE Healthcare). A monodisperse peak was obtained and assessed for purity using SDS-PAGE gel analysis, then pooled, concentrated, and exchanged into HBS buffer (10 mM Hepes [pH 7.3], 140 mM NaCl). Purified full-length zymogen and active forms of C1r were obtained from Complement Technology, Inc. Multiple sequence alignments were generated using Clustal Omega (74 (link)).
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