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Noda Institute for Scientific Research GRANT2013 Research Grant

Development of an innovative protein production systemusing second-generation mRNA export proteins created bydomain engineeringSeiji MasudaDivision of Integrated Life Science, Graduate School of Biostudies, Kyoto University

Research aimsProteins intended for use in clinics have to be producedin mammalian cells because accurate post-translationalmodifications such as glycosylation, adequate disulfidebonds, acylation, and phosphorylation are essential for theiractivity in the human body. Proteins, peptides, their analogs, and drugs that contain such components producedusing recombinant protein technology are called biotechnology-based drugs. These drugs exist in various forms;their protein-based forms include recombinant proteinswhose sequence is originally derived from an endogenousprotein and a genetically modified antibody, the nucleicacid-based forms include an aptamer and a peptide-basedform. The global sale of protein-based biomedicinesreached 86.8 billion in 2007 and an estimated 160.1 billionin 2014.
Unlike the existing drugs formed by low-molecularweight molecules, biotechnology-based drugs are known tohave specific effects on cancer and rheumatism.1, 2) Around60% of the biotechnology-based recombinant proteins currently used in the clinics are produced in CHO cells. However, protein production in CHO cells is more expensivethan that in bacteria.3) Generally, a strong promoter like theCMV promoter supports the transcription and protein production in mammalian cells. This promoter efficiently transcribes pre-mRNA from the template DNA and achieveshigh rates of protein production. Recent studies have led tothe whole genome sequencing of various species, includinghumans and bacteria, and these updates make it difficult toobtain better promoters than the CMV promoter. In addition, several attempts have been made to produce proteinseffectively in translation process.4) In the present study, anmRNA export process5) was created to establish a novelprotein production system in mammalian cells (Fig. 1).
MethodsA novel expression system, which efficiently exports aspecific mRNA to the cytosol using the MS2 binding siteand MS2 coat protein, was developed. Two expressionplasmids were constructed: one containing the luciferasegene followed by the MS2 binding sites, and the other expressing MS2 coat protein fused with Tap, an mRNA ex-Fig. 1. Schematic representation of gene expression in higher eukaryotes

Fig. 2.

Export of mRNA by MS2-Tap

porter. When MS2-Tap is expressed, it binds specifically tothe MS2 binding sites and efficiently exports its mRNA bymeans of Tap. In this way, mRNAs containing the MS2binding site can be specifically exported into the cytosol(Fig. 2).
Cells (CHO, HeLa, and HEK293) were seeded in 12well plates and cultured for 24 h. Plasmids were transfected42into the cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The transfectionefficiency was standardized with the plasmid phRL-TK expressing renilla luciferase. After culturing for 24 h, the cellswere lysed and the luciferase activity was measured using aLuciferase Assay Kit (Promega), according to the manufacturer’s instructions.
ResultsWhen the plasmid containing the MS2 binding sites wasco-transfected with that expressing MS2-Tap, the luciferaseactivity was enhanced, compared to co-transfections with acontrol MS2-GFP plasmid. In particular, the luciferase activity was enhanced 5.2 fold, 2.5 fold, and 2.5 fold in theHeLa, HEK293, and CHO cells, respectively. These resultsindicate that the protein expression system containing MS2binding sites and MS2-Tap fusion protein achieved an efficient increase in the luciferase activity in all the cells examined. Notably, the number of MS2 binding sites can changethe efficiency of the luciferase activity.
ConclusionsLuciferase activity was efficiently increased by the pro-tein expression system containing MS2 binding sites andMS2-Tap fusion protein. CHO and HEK293 are the mostfrequently used mammalian cell lines in protein production.
Our evidence suggests that enhancing mRNA export canserve as a useful strategy for protein production in mammalian cells. In the future, additional research is required todevelop an optimum combination of the MS2 binding sitesand the amount of MS2-Tap, for increasing the efficiencyof protein production in cells.
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