High-Level Expression in Escherichia coli and Purification of the Membrane-Bound Form of Cytochrome b5
| Autor: | Scott B. Mulrooney, Lucy Waskell |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Expression vector
Cytochrome b Membrane Proteins Intracellular Membranes Biology Chromatography Agarose medicine.disease_cause Molecular biology Recombinant Proteins chemistry.chemical_compound Cytochromes b5 Biochemistry chemistry Cell culture Cytochrome b5 Escherichia coli medicine Animals Agarose Electrophoresis Polyacrylamide Gel Rabbits Polyacrylamide gel electrophoresis Heme Biotechnology |
| Zdroj: | Protein Expression and Purification. 19:173-178 |
| ISSN: | 1046-5928 |
| Popis: | Expression of the membrane-bound form of rabbit cytochrome b(5) in Escherichia coli has been significantly improved through the use of the T7 expression vector pLW01 (A. Bridges, L. Gruenke, Y.-T. Chang, I. Vakser, G. Loew, and L. Waskell, 1998, J. Biol. Chem. 273, 17036-17049) in conjunction with strain C41(DE3) (B. Miroux and J. Walker, 1996, J. Mol. Biol. 260, 289-298). Cell cultures expressing the cytochrome b(5) contained an average of 820 mg/liter of culture and reached peak levels as high as 1100 mg/liter when higher antibiotic concentrations were used. Maximal levels were obtained from cultures when expression was induced with 10 microM IPTG. Approximately 90% of the cytochrome b(5) was expressed as apoprotein which was reconstituted by addition of exogenous heme. The cytochrome b(5) was purified from detergent-solubilized bacterial membranes using anion-exchange chromatography on DEAE-Sepharose followed by size-exclusion chromatography on Superdex-75. Purification of cytochrome b(5) from a 500-ml culture yielded 121 mg of protein which had a specific content of 50 nmol of heme per milligram of protein with an overall recovery of 35%. The final cytochrome b(5) was free of any detectable contaminants when analyzed by SDS-PAGE. |
| Databáze: | OpenAIRE |
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