Translation enhancement by a Dictyostelium gene sequence in Escherichia coli

Abstract

Methods for heterologous protein production in Escherichia coli have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed "translation enhancement by a Dictyostelium gene sequence" ("TED") in E. coli. We found that protein expression was increased when a gene sequence of Dictyostelium discoideum was placed upstream of the Shine-Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was mlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3' end of the mlcR gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.

Engineering the Ultrasensitive Visual Whole-Cell Biosensors by Evolved MerR and 5′ UTR for Detection of Ultratrace Mercury

“…As an essential secondary structure of RNA, the stem-loop structure of 5′ UTR can facilitate protein synthesis by guiding the subsequent activities such as RNA folding, protecting the structural stability of mRNA, and providing recognition sites for RNA-binding proteins . To understand the influence of 5′ UTR on the GFP output during these experiments, we analyzed the RNA structure by RNAfold .…”

An improved molecular tool for screening bacterial colonies using GFP expression enhanced by aDictyosteliumsequence

“…We developed a GFP expression vector in combination with TED without any adverse effects such as growth rate (14). In this system, gfp, which is placed downstream of the 25-bp fragment of the 3' end of mlcR (mlcR25) and the SD sequence, is expressed under control of the lac promoter, but IPTG is not necessary.…”

“…Here, we present a tractable tool for bacterial colony screening by GFP fluorescence based on a system called TED that enhances protein expression using a gene sequence of the social amoeba Dictyostelium discoideum in E. coli (14). In our previous work, insertion of a Dictyostelium gene sequence (e.g., mlcR) upstream of the GFP gene can spontaneously induce visible levels of GFP expression without the addition of IPTG.…”

Translation Enhancement by a Short Nucleotide Insertion at 5′UTR: Application to an In Vitro Cell-Free System and a Photosynthetic Bacterium

“…Therefore, while the simplest approach is to identify a promoter with enhanced activation, attempts are also being made to optimize the upstream sequence of the SD sequence [12]. Recently, we reported that the insertion of a gene sequence from the eukaryotic cellular slime mold Dictyostelium discoideum (e.g., mlcR encoding myosin regulatory light chain) upstream of the SD sequence increased protein production in E. coli [13]. We named this phenomenon Translation Enhancement by a Dictyostelium gene sequence (TED).…”

“…Replacing the T7 phi10 [14] in the pET vector, a typical vector for protein production, with mlcR25 can further increase production. An interesting aspect of TED used for mlcR25 is that sufficient fluorescence emitted from the green fluorescent protein (GFP), with low levels of transcription leakage from the lac promoter, was visually observed even in E. coli cells [13]. This suggests that TED may have a positive effect on translation rather than transcription.…”