Improved Tol2 transposon-based system for efficient protein production Su Young Hwang and Joon Tae Park* Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, Korea (A) Tol2 transposase mRNA was synthesized using a linearized helper vector as a template. (B) Helper vector was designed where CMV promoter-driven Tol2 transposase gene. (C) TP was designed where Tol2 right ITR and left ITR flanked CMV promoter-driven luciferase gene. (D) non-TP was designed to The establishment of mammalian cell lines with high productivity is the most
important criterion contain only CMV promoter-driven luciferase gene. (E) Comparison of protein productivity among cells in the field of biopharmaceutics. Transposon-based expression system has been developed as transfected with non-TP, TP with helper vector and TP with Tol2 transposase mRNAs. effective way to increase protein productivity. Here, we established the improved Tol2 transposon system to efficiently incorporate transgene into genome and subsequently increase protein productivity. Tol2 transposase mRNA improved the efficiency of transgene integration and protein production in Tol2
transposon-based expression systems, while preventing re- mobilization and re-integration of transposon vector. The transposon vector containing minimum cis-sequences essential for transposition (mini-TP) also served as one of the efficient means to increase protein productivity through enhancing transgene integration. Furthermore, inhibition of DNA methylation in mini-TP improved protein productivity, indicating that it is an alternative way to increase protein productivity in cells transfected with the optimal mini-TP condition. Taken Figure 2. The underlying mechanism for how Tol2
transposase mRNA improves together, our results provide new strategies to improve the Tol2 transposon-based transgene protein productivity. expression. These strategies will be applicable to produce therapeutic proteins and open new (A) Comparison of transgene integration copy numbers. (B) Western blot analysis. (C) The effect of avenues in biopharmaceutics. methylation inhibition on Tol2 transposase mRNA-based expression system. 5-AzaC and TSA were used as DNA methylation (D) Comparison of protein productivity between single cell clones. (E) The volume Keywords: Tol2 transposon system, Tol2
transposase mRNA, mini-TP, methylation protein productivity (Qp) over time was compared using single cell clones with circles shown in Fig. 2E. inhibitors Mammalian cells are the main host for protein production due to their ability to perform post- translational modifications, of which Chinese hamster ovary (CHO) cells are the most widely used cell lines (Hunter, Yuan, Vavilala, & Fox, 2019). However, mammalian cells exhibit low protein productivity, which is a major hurdle to overcome (Owczarek, Gerszberg, & Hnatuszko- Konka, 2019). Transposon system is a genetic element that mobilizes and
integrates transgene in the genome (Balasubramanian, Rajendra, Baldi, Hacker, & Wurm, 2016). Several transposon systems including sleep beauty (SB), Tol2 and piggyBac (PB) have been explored Figure 3. Role of mini-TP on Tol2 transposon-based expression system. for efficient protein production in mammalian cells and are known to enable similar protein (A) mini-TP contains a minimum cis-sequence for the Tol2 ITR consisting of 200 bp in the left ITR and productivity (Balasubramanian, Rajendra, et al., 2016). Since the insertion size of the 150 bp in the right ITR. (B) Comparison of protein
productivity among cells transfected with non-TP, TP therapeutic antibody is usually 6kb or more, transposon system capable of carrying large with Tol2 transposase mRNAs and mini-TP with Tol2 transposase mRNAs. transgenes is preferred (Balciunas et al., 2006). Tol2 transposon can integrate up to 10 kb of transgene without significantly reducing its transposition activity (Balciunas et al., 2006). In most transposon systems, transposases were introduced as a plasmid form (Muñoz-López & García-Pérez, 2010). However, persistence of transposase as episomal DNA results in persistent expression of
the transposase (Bire et al., 2013). This in turn can lead to subsequent transposon cleavage and reintegration, thus increasing potential damage to the chromosome (Bire et al., 2013). Given these findings, the use of transposase as a plasmid form has been questioned; thus, there is a need for more effective strategy to introduce transposase into cells (Bire et al., 2013; Wilber et al., 2006). Transposase promotes DNA cleavage and reintegration reactions through recognizing ITR sequence of transposon (Muñoz-López & García-Pérez, 2010). Tol2 ITRs have cis-sequences Figure 4. The underlying
mechanism for how mini-TP improves protein productivity. essential for transposition (Urasaki, Morvan, & Kawakami, 2006). The left and right ITRs are (A) Comparison of transgene integration copy numbers. (B) Western blot analysis. (C) The effect of 517bp and 536bp, respectively (Urasaki et al., 2006). The minimum cis-sequence for Tol2 ITRs methylation inhibition on Tol2 transposase mRNA-based expression system. 5-AzaC and TSA were used as DNA methylation (D) Comparison of protein productivity between single cell clones. (mini-Tol2) consisted of 200bp in the left ITR and 150bp in the right ITR
(Urasaki et al., 2006). The value of mini-TP has been strengthened by results showing that mini-TP are capable of transposition without reducing efficiency (Urasaki et al., 2006). Therefore, it would be desirable to test the effectiveness of mini-TP for transgene expression compared to full-length Tol2 ITR. Tol2 transposase mRNA could be used as a tool to improve protein productivity in In this study, we aimed to evaluate whether Tol2 transposase mRNA could be used to replace the transposon system the plasmid encoding Tol2 transposase in mammalian cells, and to test its effect on protein
mini-TP may improve our knowledge of the mechanisms leading to protein productivity. Furthermore, we evaluated the use of the core part of the Tol2 ITRs for productivity through enhancing integration and more reactive with DNA transposon-mediated transgene expression. Here, we report the utility of these new strategies methylation inhibitors. to improve the Tol2 transposon-mediated transgene expression system. This strategy to improve conventional transposon system might be applicable to produce therapeutic proteins in biopharmaceutics. Balasubramanian, S., Rajendra, Y., Baldi, L., Hacker,
D. L., & Wurm, F. M. (2016). Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines. Biotechnol Bioeng, 113 (6), 1234-1243. Urasaki, A., Morvan, G., & Kawakami, K. (2006). Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics, 174 (2), 639-649. Muñoz-López, M., & García-Pérez, J. L. (2010). DNA transposons: nature and applications in genomics. Current genomics, 11 (2), 115-128. Iida, A., Shimada,
A., Shima, A., Takamatsu, N., Hori, H., Takeuchi, K., & Koga, A. (2006). Figure 1. Role of Tol2 transposase mRNA on Tol2 transposon-based expression Targeted reduction of the DNA methylation level with 5-azacytidine promotes excision of system. the medaka fish Tol2 transposable element. Genet Res, 87 (3), 187-193. [T. Protein modification and regulation-1] Tol2 transposon-based vector platform for the efficient protein expression Su Young Hwang¹, Joon Tae Park¹ ¹Division of Life Sciences, Incheon National University, Incheon 22012, Korea The establishment of mammalian cell lines with high
productivity is the most important criterion in the field of recombinant therapeutic protein production. Transposon system has been found to be a reliable tool that enables the increased transgene integration. In this study, we used Tol2 transposon system which requires a Tol2 transposase that is needed to insert transposon vector (TP) into the genome. To prevent the risk of genomic instability due to uncontrolled activity of Tol2 transposase in DNA vectors, we used Tol2 transposase mRNA. We found that the Tol2 transposon system significantly increased transgene expression. To further improve
low transfection efficiency due to large vector size, a mini-transposon vector (mini-TP) consisting of a minimum cis-sequence for Tol2 transposon was designed. We found that the mini-TP exhibits the same activity as the original TP. Furthermore, inhibition of DNA methylation prevented gene silencing that may occur in the Tol2 transposon-mediated system. Taken together, our results are the first report to show that the use of the Tol2 transposon system with Tol2 transposase mRNA improved protein productivity. Furthermore, the potential use of mini-TP in the biopharmaceutical industry was
evidenced by similar productivity to traditional TP. “Our findings indicate that the quality control of proteasomes is essentially mediated by aggresomal sequestration and subsequent autophagic degradation in mammalian cells.” Quality Control of Mammalian Proteasome via Figure 1. Inactive proteasomes are accumulated in the aggresome Figure 2. Inactive proteasomes are transported to the aggresome through HDAC6-mediated retrograde transportation Autophagy Won Hoon Choi, Tae-rim Oh and Min Jae Lee * Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine
Abstract The 26S proteasome is a self-compartmentized protease complex with its crucial function in protein quality control. Multiple layers of regulatory systems have been identified to elaborately modulate proteasome activity for hydrolysis of polyubiquitinated proteins. However, the destruction mechanism of mammalian proteasomes responding cellular environments has been relatively poorly understood. Here, we describe that inactive 26S proteasomes are sequestered into the insoluble aggresome, a large perinuclear inclusion, via histone deacetylase 6 (HDAC6)-mediated retrograde transport. The
proteasomes were colocalized with the autophagic receptor p62/SQSTM1 and cleared through a selective macroautophagic process. Chemical and genetic inhibitions of autophagy resulted in elevated levels of proteasomes in insoluble fraction and more scattered puncta in cytoplasm, indicating that the proteaphagy is biochemically linked to aggresomal segregation. When the cells were replenished with inhibitor-free media, the aggresomal inclusion became gradually smaller and disappeared. Structural changes, association of diverse proteins, and polyubiquitination on different subunits appeared to be
involved in the targeting Figure 3. Autophagic degradation of inactive proteasomes in the aggresome mechanism of the inactive proteasome to the aggresome. These data indicate that both aggresomal sequestration and autophagic clearance are the essential process of the proteasome quality control to get rid of nonfunctional proteasomes. Figure 4. Proteasome inhibitor treatment led to significant transcriptional upregulation of proteasome subunits Results The inactive 26S proteasome can be sequestrated in the perinuclear aggresome (Figure 1) through retrograde transport mediated by the
HDAC6-dynein complex (Figure 2). To approve the fate of inactive proteasome accumulated in the aggresome, MG132 contained media was replenished with proteasome inhibitor-free media or autophagy inhibitor contained media. Consequently, the proteasome-containing aggresome was partially broken down and gradually reduced in size during the MG132 wash-out using Figure 6. Direct ubiquitination on inactive proteasome subunits normal media. On the other hand, MG132 wash-out progression in autophagy inhibition condition showed that the aggresome near the MTOC was split in part (Figure 3). The long-term
treatment of mild proteasome inhibitors increases the
