Structure-function studies on bacteriorhodopsin. IV. Purification and renaturation of bacterio-opsin polypeptide expressed in Escherichia coli.

M. S. Braiman, L. J. Stern, B. H. Chao, H. G. Khorana

Research output: Contribution to journalArticle

97 Scopus citations

Abstract

Expression of the bacterio-opsin gene in Escherichia coli has been described in the accompanying papers. We now describe rapid and efficient methods for the purification of the E. coli-expressed bacterio-opsin. Bacterio-opsin can be extracted from E. coli membranes in a denatured form by using an organic solvent containing chloroform, methanol, water, and triethylamine. The bacterio-opsin, enriched to 30-50% in the extract, can be further purified to 90% by ion-exchange chromatography on DEAE-Trisacryl or hydroxylapatite chromatography in organic solvents or by preparative sodium dodecyl sulfate gel electrophoresis. In appropriate aqueous phospholipid/detergent mixtures, up to 80% of purified protein refolds and binds retinal covalently to regenerate the bacteriorhodopsin chromophore. When reconstituted into phospholipid vesicles, bacteriorhodopsin from E. coli shows the expected proton pumping activity in response to illumination.

Original languageEnglish (US)
Pages (from-to)9271-9276
Number of pages6
JournalJournal of Biological Chemistry
Volume262
Issue number19
StatePublished - Jul 5 1987

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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