Purication and crystallization of Complement C3b Abdul Ajees Abdul Salam ( [email protected] ) Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham AL, 35294 K. Gunasekaran Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham AL, 35294 John E. Volanakis Department of Medicine, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham AL, 35294 S.V.L. Narayana Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham AL, 35294 Girish J. Kotwal Division of Medical Virology, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, HSC, Cape Town, South Africa, 7925 H.M. Krishna Murthy Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham AL, 35294 Method Article Keywords: C3b, N-Acetyl-L-Threonine, X-ray crystallography Posted Date: December 15th, 2006 DOI: https://doi.org/10.1038/nprot.2006.416 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/3 Abstract Introduction The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to pathogenesis of immunologic and inammatory diseases. Complement-component 3 \(C3) occupies a central position because of the manifold biologic activities of its activation fragments, including the major fragment, C3b, which anchors assembly of convertases effecting C3 and C5 activation. C3 is activated to C3b by proteolysis of its anaphylatoxin domain \(ANA), by either of two C3-convertases, activating a relatively stable thioester bond, leading to covalent attachment of C3b to cell-or protein-surface hydroxyl groups through trans-esterication. Cleavage/activation of C3 exposes binding sites for factors B, H, and I, properdin \(P), decay accelerating factor \(DAF, CD55), membrane cofactor protein \(MCP, CD46), complement receptor 1 \(CR1, CD35), and viral molecules such as vaccinia-virus complement-control protein \(VCP)4. C3b associates with these molecules in different congurations forming complexes, mediating activation, amplication and regulation of the complement response. Here, we present the purication and crystallization steps for C3b Reagents EDTA, KH2PO4, Benzamidine-Hcl, Trypsin, Soybean Trypsin Inhibitor, N-Acetyl-L-Threonine, Tris, NaCl, LiCl and PEG6000 Equipment DEAE and Blue sepharose anity chromatography Procedure **C3 Purication:** Step 1: Isolate Complement factor C3 by PEG precipitation from EDTA-treated human blood plasma and purify by DEAE and blue sepharose anity chromatography \(Smith, S.A., _et al._, Biochim Biophys Acta, **1650**, 30-39, 2003). **C3b preparation:** Step 2: Prepare puried C3b by performing limited trypsin digestion of C3 with sequencing grade trypsin \(Roche). Perform the cleavage in the presence of N-Acetlyl-L-Threonine \(AcT) to provide a nucleophile for covalent attachment of the side chain carbonyl of Gln991. Activate the internal thioester \(Law, A.S.K. & Dodds, A.W. Protein Science, **6**, 263-274, 1997) with 5% trypsin \(w/w) for 2 min at 37°C, before adding 5% \(w/w) soybean trypsin inhibitor \(SBTI) \(Sigma) to stop the reaction. Step 3: Remove trypsin and SBTI by perfusion chromatography using a BioCAD 20HQ column. Step 4: Visualize C3 and C3b by SDS-PAGE \(4–12% gel) with Coomassie blue staining. Step 5: Conrm esterication via mass spectroscopic analysis on tryptic digests of modied C3b. Step 6: Store modied protein at a concentration of 7.7 mg/ml in 20mM TRIS, pH 7.5 and a protein inhibitor cocktail \(1M KH2PO4, 0.2 M EDTA, 0.2 M Benzamidine-HCl and 1mM Page 2/3 PmSF). **Crystallization:** Step 7: Obtain crystals through vapor diffusion from 2 μl of the protein solution mixed with an equal volume of well solution. Wells should contain 200 mM TRIS, pH 7.5, 100 mM NaCl, 20 mM LiCl and 15% PEG6000. Timing 7 days Critical Steps Although crystals could also be grown with C3b prepared without AcT, they did not diffract beyond 6.5 Å. AcT modication improved diffraction to 2.3 Å. References 1. Smith, S.A., _et al._, _Biochim Biophys Acta_, **1650**, 30-39, 2003. 2. H.D. Gresham, D.F. Matthews, F.M. Grin Jr., _Anal. Biochem._ **154**, 454–459, 1986. 3. B.F. Tack, R.A. Harrison, J. Janatova, M.L. Thomas, J.W. Prahl, _Proc. Natl. Acad. Sci. U. S. A._ **77**, 5764– 5768, 1980. 4. Law, A.S.K. & Dodds, A.W. _Protein Science_, **6**, 263-274, 1997 Page 3/3.