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Improvement of Sleeping Beauty Transposon System Enabling Efficient and Stable Protein Production
이윤행,박지윤,송은선,이하늘,국명욱,주정현,노형민,박준태 한국생물공학회 2022 Biotechnology and Bioprocess Engineering Vol.27 No.3
The most essential goal in the field of biopharmaceuticals is to develop cell lines with higher protein yields. To this goal, the Sleeping Beauty (SB) transposonbased expression system has been developed as a powerful tool for increasing protein productivity. However, SB transposon system has fallen short of expectation in terms of the efficiency and stability of protein production, limiting its applicability to large-scale production of recombinant proteins. Here, we propose a novel strategy to increase the efficiency and stability of protein production, through modification of the traditional SB transposon vector. Adding a pair of inverted terminal repeats (ITRs) next to existing ITRs (i.e., double-ITRs) significantly increased the efficiency of transgene integration, resulting in high-yield and sustained protein production. Furthermore, double- ITRs responded more favorably to DNA methylation inhibitors in terms of protein yield, implying that using double-ITRs with DNA methylation inhibitors may be effective in increasing protein productivity. Taken together, our study introduces a new vector platform that is applicable to high-yield and sustained protein production, and will open new avenues in the field of biopharmaceuticals.
이윤행,이건주,김학희 한국공업화학회 2002 응용화학 Vol.6 No.2
Activated carbon was prepared from black sugar by chemical activation withNaOH. The preparation process was carried out by carbonization chemicalactivation washing, and dry The effect of the carbonization temperature and chemical ratio on the production cost is so strong that in the region of influence,it is recommended to use possible low temperature and chemical ratio. Optimalcarbonization temperature and time were found to be 500℃ and 1hrreapectively. N_2-BET surface areas were measured as about 478∼574m^2/g with100∼300wt% chemical ratio of NaOH. The use of activated carbon made fromblack sugar allows for the efficient removal of oranic pollutants in waste water.
Improvement of Tol2 Transposon System Enabling Efficient Protein Production in CHO Cells
황수영,이윤행,국명욱,김재원,오세경,박준태 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.5
Establishment of mammalian cell lines with high protein productivity is an important object in the field of biopharmaceutics. Toward this end, Tol2 transposonbased expression systems have been developed as effective means to facilitate protein productivity. Here, we proposed novel strategies to improve conventional Tol2 transposon systems. The use of Tol2 transposase mRNA as a helper vector improved the efficiency of transgene integration and protein production. Moreover, the use of the Tol2 transposon vector containing the minimum cis-sequences essential for transposition (mini-TP) also served as one of the efficient means to generate recombinant cells that enable higher protein production. Furthermore, mini-TP showed a more beneficial response to DNA methylation inhibitors, suggesting that the use of mini-TP with DNA methylation inhibitors could be used as a means of commercial production. Taken together, our results provide effective strategies to improve the Tol2 transposon-based expression system. These strategies will be applicable to the production of therapeutic proteins and open new avenues in biopharmaceutical research.
Cre/Lox-based RMCE for Site-specific Integration in CHO Cells
김재원,이윤행,국명욱,황수영,권형욱,박준태 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.5
Traditional cell line development is based on random genomic integration of transgenes. Random integration leads to unpredictable expression and results in clonal heterogeneity requiring a tedious screening procedure. Therefore, a new strategy is needed to establish clones that exhibit stable transgene expression. Here, we performed CRISPR/Cas9-mediated site-specific integration (SSI) to incorporate a landing pad (LP; containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and strong expression. Site-specific integration of LP on Fer1L4 was demonstrated by sequencing results representing the swapped sequences in mCherry-expressing cells. We then performed Cre/Lox-based recombinase-mediated cassette exchange (RMCE) to exchange LP with a targeting vector (TV; containing GFP) in clones established by CRISPR/ Cas9-mediated SSI. The success of Cre/Lox-based RMCE was evidenced by sequencing results representing the swapped sequences in GFP-expressing cells. Furthermore, the swapped clones expressing GFP was enriched up to 80%, indicating that the efficiency of Cre/Lox-based RMCE would be sufficient to swap pre-existing cassettes with gene-of-interest (GOI). Taken together, our study provides a new platform for Cre/Lox-based RMCE to iteratively establish stable clones from existing ones previously established by SSI at a genomic hotspot.
Development of a Quadruped Walking Robot AiDIN-III using Biologically Inspired Kinematic Analysis
구익모,Duc Trong Tran,이윤행,문형필,구자춘,박상덕,최혁렬 제어·로봇·시스템학회 2013 International Journal of Control, Automation, and Vol.11 No.6
This paper presents a bio-inspired mechanism design for a quadruped walking robot. The approach is derived from the observation on the behaviors of quadruped locomotion, skeletal structure, and the study on the stability of walking based on morphological analysis. In the first, we define the design parameters such as the dimensions of the body and limbs, the center of mass position, and locomotion mechanisms based on surveys on the literatures from biologists. Then, by using the parameters, we propose an useful framework for determining the design parameters of a quadruped walking robot. For implementations, we manufacture a dog-type self-contained quadruped walking robot, named AiDIN-III (Artificial DIgitigrade for Natural Environment version III) and the effectiveness of the proposed idea is validated via experimental works.