Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied for their structure and function. These retinal-binding proteins can be divided into two types. Type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. The two primary functions of type I rhodopsin are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin with a specific function of photosensory transduction. ASR is expressed at moderate levels in Escherichia coli, but its expression level is lower compared to the general green light absorbing proteorhodopsin (GPR). In this study, full-length ASR was used to test the influence of codon usage on expression E. coli. Seven amino acids at the N-terminal region of ASR after the Met start codon were changed randomly using designed primers, which allowed for 8192 different nucleotide combinations. The codon changes were screened for the preferable codons that resulted in higher expression yield. Among the 57 selected mutations, 24 color-enhanced E. coli colonies contained ASR proteins, and they expressed ASR at a higher level than the bacteria with wild-type ASR codon usage. This result strongly suggests that the specific codon usage of only the N-terminal portion of a protein can increase the expression level of the entire protein.
Keywords: 7-Transmembrane; All-trans retinal; Codon-usage; Protein expression; Rhodopsin.
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