Tsogbadrakh Otgontuya, Choi Ah Reum, Jung Kwang-Hwan
Institute of Chemistry and Chemical Technology, Mongolian Academy of Sciences, Ulaanbaatar 51, Mongolia.
Department of Life Science and Institute of Biological Interfaces, Sogang University, Seoul, 121-742, South Korea.
Protein Expr Purif. 2018 Nov;151:1-8. doi: 10.1016/j.pep.2018.05.011. Epub 2018 May 22.
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.
微生物视紫红质是著名的七跨膜蛋白，其结构和功能已得到广泛研究。这些视网膜结合蛋白可分为两类。第一类是微生物视紫红质，第二类（视觉色素）主要在哺乳动物眼睛中表达。第一类视紫红质的两个主要功能是离子泵浦活性和感官转导。鱼腥藻感光视紫红质（ASR）是一种具有光感受转导特定功能的微生物视紫红质。ASR在大肠杆菌中适度表达，但其表达水平与一般吸收绿光的视紫质（GPR）相比更低。在本研究中，使用全长ASR来测试密码子使用对其在大肠杆菌中表达的影响。使用设计的引物随机改变ASR在甲硫氨酸起始密码子之后N端区域的七个氨基酸，这产生了8192种不同的核苷酸组合。筛选密码子变化以找出导致更高表达产量的优选密码子。在所选的57个突变中，24个颜色增强的大肠杆菌菌落含有ASR蛋白，并且它们表达ASR的水平高于具有野生型ASR密码子使用的细菌。这一结果有力地表明，仅蛋白质N端部分的特定密码子使用就可以提高整个蛋白质的表达水平。
