TY - JOUR
T1 - A unified method for purification of basic proteins
AU - Adhikari, Sanjay
AU - Manthena, Praveen Varma
AU - Sajwan, Kamal
AU - Kota, Krishna Kiran
AU - Roy, Rabindra
N1 - Funding Information:
We thank Prof. Sankar Mitra and Dr. Tapas Hazra of UTMB, Galveston, Texas, for providing the mAPE1 and hOGG1 cDNA. We also thank Prof. Eliot Rosen of Georgetown University for GST-BRCA1 cDNA. We thank Dr. Amrita Cheema for proteomics experiments performed at the Proteomics and Metabolomics Shared Resource of the Lombardi Comprehensive Cancer Center. We thank Protein Core facility, UTMB, Texas, for amino acid sequence analysis. We also thank Mr. Cliff Chung and Ms. Karen Howenstein for expert editorial help. The work was supported in part by NIH Grants RO1 CA 92306 and RO1 CA 113447 awarded to R.R.
PY - 2010/5
Y1 - 2010/5
N2 - Protein purification is still very empirical, and a unified method for purifying proteins without an affinity tag is not available yet. In the postgenomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied for purifying any recombinant basic protein from Escherichia coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (p. I) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the p. I determined theoretically, apparently all recombinant basic proteins (at least p. I-1 ≥ 6.94) may be purified from E. coli in a single step using a cation-exchanger resin, SP-Sepharose, and a selected buffer pH, depending on the p. I of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a p. I of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step.
AB - Protein purification is still very empirical, and a unified method for purifying proteins without an affinity tag is not available yet. In the postgenomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied for purifying any recombinant basic protein from Escherichia coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (p. I) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the p. I determined theoretically, apparently all recombinant basic proteins (at least p. I-1 ≥ 6.94) may be purified from E. coli in a single step using a cation-exchanger resin, SP-Sepharose, and a selected buffer pH, depending on the p. I of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a p. I of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step.
KW - Basic protein
KW - Ion-exchange chromatography
KW - Protein purification
KW - Single step
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U2 - 10.1016/j.ab.2010.01.011
DO - 10.1016/j.ab.2010.01.011
M3 - Article
C2 - 20109435
AN - SCOPUS:77950691139
SN - 0003-2697
VL - 400
SP - 203
EP - 206
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -