Purification and crystallization of Complement C3b
Lab/Group: Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham
Related Journal & Article Information
Journal: Nature
Article Title: The structure of complement C3b provides insights into complement activation and regulation
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 inflammatory 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-esterification. 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 configurations forming complexes, mediating activation, amplification and regulation of the complement response. Here, we present the purification and crystallization steps for C3b
Materials
Reagents
EDTA, KH2PO4, Benzamidine-Hcl, Trypsin, Soybean Trypsin Inhibitor, N-Acetyl-L-Threonine, Tris, NaCl, LiCl and PEG6000
Equipment
DEAE and Blue sepharose affinity chromatography
Procedure
C3 Purification:
Step 1: Isolate Complement factor C3 by PEG precipitation from EDTA-treated human blood plasma and purify by DEAE and blue sepharose affinity chromatography (Smith, S.A., et al., Biochim Biophys Acta, 1650, 30-39, 2003).
C3b preparation:
Step 2: Prepare purified 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: Confirm esterification via mass spectroscopic analysis on tryptic digests of modified C3b.
Step 6: Store modified 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 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.
Troubleshooting
Critical Steps
Although crystals could also be grown with C3b prepared without AcT, they did not diffract beyond 6.5 Å. AcT modification improved diffraction to 2.3 Å.
Anticipated Results
References
1. Smith, S.A., et al., Biochim Biophys Acta, 1650, 30-39, 2003.
2. H.D. Gresham, D.F. Matthews, F.M. Griffin 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
Acknowledgements
Keywords
C3b, N-Acetyl-L-Threonine