Email this article 
Modified CNBr Cleavage Protocol for Efficient Separation of Met-Ser Containing OmpX-Om14 Membrane Protein Fusion
(*) Corresponding author
DOI's assignment:
the author of the article can submit here a request for assignment of a DOI number to this resource!
Cost of the service: euros 10,00 (for a DOI)
Abstract
Cleavage efficiencies of cyanogen bromide (CNBr) are drastically reduced in sequences wherein the methionine residue is succeeded by serine or threonine. Solubility issues associated especially with membrane proteins contribute further to reduced cleavage at Met-Xxx sites. Moreover, prolonged incubations of several hours to days, in highly acidic reaction conditions required for CNBr cleavage, often lead to protein degradation. To circumvent these problems, we have developed a protocol for CNBr cleavage of protein fusions, which is particularly useful for Met-Ser sequences. This protocol facilitates enhanced cleavage efficiencies in short incubation times of 1-2 hours. It incorporates up to 40% acetonitrile in 8M urea, or trace amounts of acetonitrile in 6M guanidine hydrochloride, in the final reaction. We demonstrate the successful application of this protocol for the Met-Ser containing membrane protein fusion OmpX-Om14. Cleavage of the Om14 (the yeast mitochondrial outer membrane protein) from E. coli OmpX (Outer membrane protein X) has provided yields of up to 70% product without detectable degradation levels. The direct use of the cleavage reaction for the rapid and cost-effective purification of Om14 has been shown. Using a database analysis, we discuss the importance of this protocol for several membrane proteins that have Met-Ser sequences
Copyright © 2012 Praise Worthy Prize - All rights reserved.
Keywords
References
J. Hoppe, H. A. Weich, W. Eichner, Preparation of Biologically Active Platelet-Derived Growth Factor Type Bb from a Fusion Protein Expressed in Escherichia coli, Biochemistry, Volume 28, (Issue 7), April 1989, Pages 2956-2960.
J. C. Rodriguez, L. Wong, P. A. Jennings, The Solvent in CNBr Cleavage Reactions Determines the Fragmentation Efficiency of Ketosteroid Isomerase Fusion Proteins Used in the Production of Recombinant Peptides, Protein Expression and Purification, Volume 28, (Issue 2), April 2003, Pages 224-231.
A. Mitra, K. S. Chakrabarti, M. S. Shahul Hameed, K. V. Srinivas, G. Senthil Kumar, S. P. Sarma, High Level Expression of Peptides and Proteins Using Cytochrome B5 as a Fusion Host, Protein Expression and Purification, Volume 41, (Issue 1), May 2005, Pages 84-97.
T. J. Park, S. S. Choi, G. A. Gang, Y. Kim, High-Level Expression and Purification of the Second Transmembrane Domain of Wild-Type and Mutant Human Melanocortin-4 Receptor for Solid-State NMR Structural Studies, Protein Expression and Purification, Volume 62, (Issue 2), December 2008, Pages 139-145.
K. E. Caroccia, R. Estephan, L. S. Cohen, B. Arshava, M. Hauser, O. Zerbe, J. M. Becker, F. Naider, Expression and Biophysical Analysis of a Triple-Transmembrane Domain-Containing Fragment from a Yeast G Protein-Coupled Receptor, Biopolymers, Volume 96, (Issue 6), December 2011, Pages 757-771.
G. A. Cook, S. Stefer, S. J. Opella, Expression and Purification of the Membrane Protein P7 from Hepatitis C Virus, Biopolymers, Volume 96, (Issue 1), February 2011, Pages 32-40.
R. Kaiser, L. Metzka, Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds, Analytical Biochemistry, Volume 266, (Issue 1), January 1999, Pages 1-8.
B. J. Smith, Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds (Humana Press Inc., 2002, pp. 485-491).
E. Gross, B. Witkop, Non-enzymatic Cleavage of Peptide Bonds: The Methionine Residues in Bovine Pancreatic Ribonuclease, Journal of Biological Chemistry, Volume 237, (Issue 6), June 1962, Pages 1856-1860.
A. Kuliopulos, C. T. Walsh, Production, Purification, and Cleavage of Tandem Repeats of Recombinant Peptides, Journla of the American Chemical Society, Volume 116, (Issue 11), June 1994, Pages 4599-4607.
Y. A. Andreev, S. A. Kozlov, A. A. Vassilevski, E. V. Grishin, Cyanogen Bromide Cleavage of Proteins in Salt and Buffer Solutions, Analytical Biochemistry, Volume 407, (Issue 1), December 2010, Pages 144-146.
P. Kraft, J. Mills, E. Dratz, Mass Spectrometric Analysis of Cyanogen Bromide Fragments of Integral Membrane Proteins at the Picomole Level: Application to Rhodopsin, Analytical Biochemistry, Volume 292, (Issue 1), May 2001, Pages 76-86.
A. K. Somani, J. S. Bignon, G. B. Mills, K. A. Siminovitch, D. R. Branch, Src Kinase Activity Is Regulated by the Shp-1 Protein-Tyrosine Phosphatase, Jounal of Biological Chemistry, Volume 272, (Issue 34), August 1997, Pages 21113-21119.
R. B. Mandell, C. M. Feldherr, Identification of Two Hsp70-Related Xenopus Oocyte Proteins That Are Capable of Recycling across the Nuclear Envelope, Journal of Cell Biology, Volume 111, (Issue 5 Part 1), November 1990, Pages 1775-1783.
J. R. Rusche, K. Javaherian, C. McDanal, J. Petro, D. L. Lynn, R. Grimaila, A. Langlois, R. C. Gallo, L. O. Arthur, P. J. Fischinger, et al., Antibodies That Inhibit Fusion of Human Immunodeficiency Virus-Infected Cells Bind a 24-Amino Acid Sequence of the Viral Envelope, Gp120, Proceedings of the National Academy of Sciences U. S. A., Volume 85, (Issue 9), May 1988, Pages 3198-3202.
J. Tchou, A. P. Grollman, The Catalytic Mechanism of Fpg Protein. Evidence for a Schiff Base Intermediate and Amino Terminus Localization of the Catalytic Site, Journal of Biological Chemistry, Volume 270, (Issue 19), May 1995, Pages 11671-11677.
F. M. Assadi-Porter, S. Patry, J. L. Markley, Efficient and Rapid Protein Expression and Purification of Small High Disulfide Containing Sweet Protein Brazzein in E. coli, Protein Expression and Purification, Volume 58, (Issue 2), April 2008, Pages 263-268.
W. D. Fairlie, A. D. Uboldi, D. P. De Souza, G. J. Hemmings, N. A. Nicola, M. Baca, A Fusion Protein System for the Recombinant Production of Short Disulfide-Containing Peptides, Protein Expression and Purification, Volume 26, (Issue 1), October 2002, Pages 171-178.
S. Lauffer, K. Mabert, C. Czupalla, T. Pursche, B. Hoflack, G. Rodel, U. Krause-Buchholz, Saccharomyces cerevisiae Porin Pore Forms Complexes with Mitochondrial Outer Membrane Proteins Om14p and Om45p, Journal of Biological Chemistry, Volume 287, (Issue 21), May 2012, Pages 17447-17458.
R. Mahalakshmi, C. M. Franzin, J. Choi, F. M. Marassi, Nmr Structural Studies of the Bacterial Outer Membrane Protein Ompx in Oriented Lipid Bilayer Membranes, Biochimica et Biophysica Acta, Volume 1768, (Issue 12), December 2007, Pages 3216-3224.
A. R. Shenoy, S. S. Visweswariah, Site-Directed Mutagenesis Using a Single Mutagenic Oligonucleotide and Dpni Digestion of Template DNA, Analytical Biochemistry, Volume 319, (Issue 2), August 2003, Pages 335-336.
V. Jain, R. Saleem-Batcha, A. China, D. Chatterji, Molecular Dissection of the Mycobacterial Stringent Response Protein Rel, Protein Science, Volume 15, (Issue 6), June 2006, Pages 1449-1464.
W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, H. L. Heyneker, Single-Step Assembly of a Gene and Entire Plasmid from Large Numbers of Oligodeoxyribonucleotides, Gene, Volume 164, (Issue 1), October 1995, Pages 49-53.
R. Mahalakshmi, F. M. Marassi, Orientation of the Escherichia coli Outer Membrane Protein Ompx in Phospholipid Bilayer Membranes Determined by Solid-State NMR, Biochemistry, Volume 47, (Issue 25), June 2008, Pages 6531-6538.
G. E. Tusnady, Z. Dosztanyi, I. Simon, Pdb_Tm: Selection and Membrane Localization of Transmembrane Proteins in the Protein Data Bank, Nucleic Acids Research, Volume 33, (Database issue), January 2005, Pages D275-D278.
Refbacks
- There are currently no refbacks.
Please send any question about this web site to info@praiseworthyprize.com
Copyright © 2005-2024 Praise Worthy Prize
