Date Published: May 21, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Chang-Ye Hui, Yan Guo, Wen Zhang, Xian-Qing Huang.
Real-time quantification of recombinant proteins is important in studies on fermentation engineering, cell engineering, etc. Measurement of the expression level of heterologous proteins in bacterial fermentation broth has traditionally relied on time-consuming and labor-intensive procedures, such as polyacrylamide gel electrophoresis, immunoblot analysis, and biological activity assays. We describe a simple, fast, and high sensitive assay for detecting heterologous proteins production in bacteria either at the overall level (fluorescence spectrophotometry) or at the individual level (fluorescence microscopic image) in this study. Based on a dicistronic model, the translation of target gene in the upstream open reading frame (ORF) was coupled with the synthesis of the mCherry reporter in the downstream ORF in E. coli cells, and subsequently this demonstrated a positive correlation between the expression of target gene and mCherry. Although a time lag exists between the expression of target protein and mCherry reporter, the method described here allows facile monitoring of dynamic changes in target protein expression, relying on indirect determination of the fluorescence intensity of mCherry during fermentation in real-time models. Additionally, the performance of a single bacterial cell factory could be checked under the fluorescence microscope field.
It is a long time since Escherichia coli (E. coli) was developed as an ideal microbial cell factory for the production of recombinant proteins. The application of highly active bacteriophage RNA polymerase, especially T7 RNA polymerase, made the expression system very powerful for overproduction of recombinant proteins (Peranen et al. 1996). An E. coli-based expression system is certainly used as the preferred system, and numerous heterologous proteins of different origins have been successfully expressed in recombinant E. coli (Hui et al. 2018b; Mahalik et al. 2014).
In E. coli, RBS and other regulatory RNA sequences are essential control elements for translation initiation (Carrier and Keasling 1999; Isaacs et al. 2004). Translation initiation, the rate-limiting step of protein expression, is determined by several molecular interactions, such as the complementarity of the RBS sequence to 16S rRNA, the spacing between RBS and the start codon, and the presence of mRNA secondary structures (Salis et al. 2009). In some cases, the distance between the promoter and structural genes in natural operons is 60 bases above. The necessary elements for translation initiation including: the standby site, 16S rRNA binding site, spacing and the start codon are all included in the intergenic regions. So each gene in the polycistronic mRNA strand can be translated independently and tunably (de Smit and van Duin 2003; Jacob et al. 2005; Kudla et al. 2009).