From Left: Dr. James Chen Yong Kah, Kian Ping Chan, and Dr. Sheng-Hao Chao
Kian Ping Chan1,2,3, Yang Gao1, Jeremy Xianwei Goh1, Dewi Susanti4, Eugenia Li Ling Yeo1, Sheng-Hao Chao2,5, James Chen Yong Kah1,3
1 Department of Biomedical Engineering, National University of Singapore, Singapore
2 Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore
3 NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore
4 Faculty of Science, National University of Singapore, Singapore
5 Department of Microbiology and Immunology, National University of Singapore, Singapore
ACS Applied Materials & Interfaces 2017 9(12): 10408-10417 (Online Version)
An in vitro translation kit provides a cell-free system to synthesize proteins of interest. This system offers a useful tool to synthesize a variety of proteins, including toxic or unstable proteins, for biological research. It can also be applied for protein folding studies and functional studies by incorporation of modified or non-natural amino acids. Many companies, such as Thermo Fisher and Promega, have developed their own in vitro translation kits using lysates prepared from different types of cells and have optimized for high protein yield by adjusting for parameters such as temperature and ion concentration. It has been shown that DNA functionalized gold nanoparticles (AuNP-DNA), in which DNA oligomers are conjugated to AuNP, are capable of enhancing in vitro translation. In this study, we extended previous works by using the alternative 3’-untranslated region (UTR) of mRNAs for DNA oligomer hybridization. We designed the DNA oligomer sequences that hybridized to the 3’-UTRs of two mRNAs of interest, insulin and green fluorescent protein (GFP), and generated the specific AuNP-DNA. With the addition of respective AuNP-DNA in an in vitro translation kit, the protein yield of insulin and GFP increased up to 2-fold. Disruption of complementary hybridization between DNA oligomers and mRNAs consequently eliminated the translation enhancement. We also demonstrated that ribosomal proteins were recruited to AuNP-DNA’s surface to form a layer of protein corona around the particle. Both non-specific adsorption of ribosomes on AuNP-DNA and hybridization between AuNP-DNA and mRNA molecules are important factors that bring translation machineries into close proximity. This could reduce the recycling time of ribosomes during in vitro mRNA translation, thereby increasing the efficiency of protein synthesis. This study allowed us to betterunderstand the mechanism behind translation enhancement by AuNP-DNA, to establish guidelines for rational design of DNA oligomer sequences, and to better design the AuNP-DNA construct for optimization of in vitro protein synthesis.
Figure 1. Enhancement of in vitro protein synthesis using AuNP-DNA. . (A) Western blot results showing the amount of HA-tagged insulin (INS-HA). Cell lysates from in vitro translation kits were incubated with no AuNP (Control), AuNP (+AuNP), thymine-conjugated AuNP (+AuNP-ctDNA), and INS-HA 3’-UTR-conjugated AuNP (+AuNP-wkDNA). Actin was used as the loading control. (B) Model for in vitro mRNA translation enhancement using AuNP-DNA.
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