This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Purification and crystallization of Complement C3b
Abdul Ajees Abdul Salam
Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham AL, 35294
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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
EDTA, KH2PO4, Benzamidine-Hcl, Trypsin, Soybean Trypsin Inhibitor, N-Acetyl-L-Threonine, Tris, NaCl, LiCl and PEG6000
DEAE and Blue sepharose affinity chromatography
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.
7 days
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.
- Smith, S.A., et al., Biochim Biophys Acta, 1650, 30-39, 2003.
- H.D. Gresham, D.F. Matthews, F.M. Griffin Jr., Anal. Biochem. 154, 454–459, 1986.
- 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.
- Law, A.S.K. & Dodds, A.W. Protein Science, 6, 263-274, 1997
commented on 27 January, 2011
It should be noted that the correctness of the structure reported in the publication associated with this Protocol was called into question ( http://www.nature.com/nature/journal/v448/n7154/full/nature06102.html and authors' response http://www.nature.com/nature/journal/v448/n7154/full/nature06103.html ). Further investigation by the University of Alabama resulted in the removal of the structure from the Protein Data Bank (Statement from UAB: http://main.uab.edu/Sites/reporter/articles/71570/ ). See also Nature News report: "Fraud rocks protein community" http://www.nature.com/news/2009/091222/full/462970a.htmlThis Protocol should therefore be treated with caution.
commented on 27 January, 2011
It should be noted that the correctness of the structure reported in the publication associated with this Protocol was called into question ( http://www.nature.com/nature/journal/v448/n7154/full/nature06102.html and authors' response http://www.nature.com/nature/journal/v448/n7154/full/nature06103.html ). Further investigation by the University of Alabama resulted in the removal of the structure from the Protein Data Bank (Statement from UAB: http://main.uab.edu/Sites/reporter/articles/71570/ ). See also Nature News report: "Fraud rocks protein community" http://www.nature.com/news/2009/091222/full/462970a.htmlThis Protocol should therefore be treated with caution.
Version History
CURRENT STATUS: Posted
Version 1
Posted 14 Dec, 2006
Metrics
Comments: 2
HTML Views: ...
Associated Publications
The structure of complement C3b provides insights into complement activation and regulation
by Ajees, A.A. et al.
Nature (24 August, 2006)
Method Article
Purification and crystallization of Complement C3b
Abdul Ajees Abdul Salam
Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham AL, 35294
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 14 Dec, 2006
Metrics
Comments: 2
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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
EDTA, KH2PO4, Benzamidine-Hcl, Trypsin, Soybean Trypsin Inhibitor, N-Acetyl-L-Threonine, Tris, NaCl, LiCl and PEG6000
DEAE and Blue sepharose affinity chromatography
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.
7 days
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.
- Smith, S.A., et al., Biochim Biophys Acta, 1650, 30-39, 2003.
- H.D. Gresham, D.F. Matthews, F.M. Griffin Jr., Anal. Biochem. 154, 454–459, 1986.
- 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.
- Law, A.S.K. & Dodds, A.W. Protein Science, 6, 263-274, 1997
commented on 27 January, 2011
It should be noted that the correctness of the structure reported in the publication associated with this Protocol was called into question ( http://www.nature.com/nature/journal/v448/n7154/full/nature06102.html and authors' response http://www.nature.com/nature/journal/v448/n7154/full/nature06103.html ). Further investigation by the University of Alabama resulted in the removal of the structure from the Protein Data Bank (Statement from UAB: http://main.uab.edu/Sites/reporter/articles/71570/ ). See also Nature News report: "Fraud rocks protein community" http://www.nature.com/news/2009/091222/full/462970a.htmlThis Protocol should therefore be treated with caution.
commented on 27 January, 2011
It should be noted that the correctness of the structure reported in the publication associated with this Protocol was called into question ( http://www.nature.com/nature/journal/v448/n7154/full/nature06102.html and authors' response http://www.nature.com/nature/journal/v448/n7154/full/nature06103.html ). Further investigation by the University of Alabama resulted in the removal of the structure from the Protein Data Bank (Statement from UAB: http://main.uab.edu/Sites/reporter/articles/71570/ ). See also Nature News report: "Fraud rocks protein community" http://www.nature.com/news/2009/091222/full/462970a.htmlThis Protocol should therefore be treated with caution.
Associated Publications
The structure of complement C3b provides insights into complement activation and regulation
by Ajees, A.A. et al.
Nature (24 August, 2006)