Expression of octopus rhodopsin in Escherichia coli.
Apoproteins, having various molecular weights, of octopus rhodopsin (oRh, 455 amino acids), which is a typical transmembrane protein, were expressed in Escherichia coli with an inducible expression system using promoter phi 10 of the T7 phage. Fifteen synthetic genes (212-1,365 bp) for oRh (1) were cloned downstream from gene 10 of the T7 phage (846 bp), under the control of promoter phi 10. An expression vector for mature oRh containing no extra peptide resulting from gene 10 was also constructed. Protein productivities were mainly evaluated by ELISA using monoclonal antibodies. The expression level in E. coli of the fused oRh genes varied between 1 and 200 mg/liter of the culture medium (from approximately 0.25 to 50% of the total cell protein, respectively), depending on the fused oRh genes. The amount of mature oRh protein expressed in E. coli was approximately 0.1 to 1 mg/liter. Hydropathy index analysis of gene products showed a significant negative correlation (rho = - 0.63) between expression level of oRh gene products in E. coli and their hydrophobic characteristics. Wavelength shifting of the absorption maximum by exogenous addition of retinal to apoprotein similar to that of authentic oRh was demonstrated in the membrane fraction of E. coli expressing mature opsin.
Harada
Y
Y
Advanced Research Laboratory, Hitachi, Ltd, Saitama.
Senda
T
T
Sakamoto
T
T
Takamoto
K
K
Ishibashi
T
T
D000818:Animals; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D049831:Octopodiformes; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012243:Rhodopsin / Q000096:biosynthesis* / Q000235:genetics; D017299:Rod Opsins / Q000096:biosynthesis; D013053:Spectrophotometry
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D012243:Rhodopsin