title: Structure-function studies on bacteriorhodopsin. II. Improved expression of the bacterio-opsin gene in Escherichia coli.
abstract: The aims of this work have been to express bacterio-opsin with minimal variation from the native primary structure and to improve the level of expression in Escherichia coli. We describe the construction of plasmids in which the bacterio-opsin gene contains only an additional methionine residue at the N terminus and in which the C-terminal aspartic acid encoded in the gene has been deleted to conform to the mature protein. In attempts to improve bacterio-opsin expression, a variety of expression plasmids were constructed in which the promoters and the ribosome-binding sequences were varied. Invariably, in these plasmids, translation but not transcription of the bacterio-opsin gene was limiting. A striking increase in expression of the gene occurred when the codons for several of the N-terminal amino acids were changed to increase the A = T content. Bacterio-opsin expressed in E. coli was degraded with a half-life of 8-10 min. The addition of hydrophobic signal sequences at the N terminus increased the half-life and overall yield of the protein. Bacterio-opsin thus produced regenerated the native bacteriorhodopsin-like chromophore and carried out light-dependent proton translocation at a rate comparable to that of the native bacterio-opsin prepared from the purple membrane.
authors.forename: S S
authors.initials: SS
authors.lastname: Karnik
authors.forename: M
authors.initials: M
authors.lastname: Nassal
authors.forename: T
authors.initials: T
authors.lastname: Doi
authors.forename: E
authors.initials: E
authors.lastname: Jay
authors.forename: V
authors.initials: V
authors.lastname: Sgaramella
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D006207:Half-Life; D007700:Kinetics; D010957:Plasmids; D013329:Structure-Activity Relationship
chemical_list: D001436:Bacteriorhodopsins; C017827:bacterio-opsin