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Ahn, Jiyeon,Nam, Ky-Youb,Lee, Sae-Lo-Oom,Ryu, Hwani,Choi, Hyun Kyung,Song, Jie-Young Korean Chemical Society 2014 Bulletin of the Korean Chemical Society Vol.35 No.12
Nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates the expression of over 200 genes of antioxidant and phase II drug-metabolizing enzymes, and is highly expressed in non-small cell lung cancer (NSCLC). Nine derivatives of 4-(2-cyclohexylethoxy)aniline were designed. Our previous study demonstrated that IM3829 increases radiosensitivity of several lung cancer cells in vitro and in vivo. Here, biological effects of IM3829 derivatives (2a-2i) were evaluated. Compound 2g derivative effectively inhibits mRNA and protein expression of Nrf2 and HO-1. In addition, we observed over two fold enhancement in IR-induced cell death, from $2.90{\pm}0.22$ to $6.02{\pm}0.87$, in H1299 cancer cell-line. Among the nine derivatives, compound 2g derivative exhibited the highest enhancement of radiosensitizing effect via inhibition of Nrf2 activity.
Crystal Structure of Mesaconyl-CoA Hydratase from Methylorubrum extorquens CM4
Ahn Jae-Woo,Hong Jiyeon,Kim Kyung-Jin 한국미생물·생명공학회 2023 Journal of microbiology and biotechnology Vol.33 No.4
Methylorubrum extorquens, a facultative methylotroph, assimilates C1 compounds and accumulates poly-β-hydroxylbutyrate (PHB) as carbon and energy sources. The ethylmalonyl pathway is central to the carbon metabolism of M. extorquens, and is linked with a serine cycle and a PHB biosynthesis pathway. Understanding the ethylmalonyl pathway is vital in utilizing methylotrophs to produce value-added chemicals. In this study, we determined the crystal structure of the mesaconyl-CoA hydratase from M. extorquens (MeMeaC) that catalyzes the reversible conversion of mesaconyl-CoA to β-methylmalyl-CoA. The crystal structure of MeMeaC revealed that the enzyme belongs to the MaoC-like dehydratase domain superfamily and functions as a trimer. In our current MeMeaC structure, malic acid occupied the substrate binding site, which reveals how MeMeaC recognizes the β-methylmalyl-moiety of its substrate. The active site of the enzyme was further speculated by comparing its structure with those of other MaoC-like hydratases.
Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter
Ahn, Jungoh,Hong, Jiyeon,Park, Myongsoo,Lee, Hyeokweon,Lee, Eungyo,Kim, Chunsuk,Lee, Joohwan,Choi, Eui-sung,Jung, Joon-ki,Lee, Hongweon American Society for Microbiology 2009 Applied and environmental microbiology Vol.75 No.11
<B>ABSTRACT</B><P>To develop a functional phosphate-regulated promoter in <I>Pichia pastoris</I>, a phosphate-responsive gene, <I>PHO89</I>, which encodes a putative sodium (Na<SUP>+</SUP>)-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the <I>PHO89</I> promoter (<I>PPHO89</I>) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. <I>PPHO89</I> was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1α and the glyceraldehyde-3-phosphate dehydrogenase promoter, <I>PPHO89</I> exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple <I>PPHO89</I>-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of <I>PPHO89</I> for controlled production of recombinant proteins in <I>P. pastoris</I>.</P>
Improvement of Artificial Antibody Secretion Using Supercharged Protein
Jiyeon Park(박지연),Heeju Choi(최희주),Hyejin Lee(이혜진),Jung Hoon Ahn(안정훈) 한국생명과학회 2020 생명과학회지 Vol.30 No.5
Repebody는 비면역 글로블린 인공 항체로 저렴하고 빠르게 생산 가능한 맞춤형 항체이다. 그러나 의료용 repebody의 생산은 저수율 및 복잡한 정제 공정으로 인해 여전히 어려움을 겪고 있다. Pseudomonas fluorescens의 ABC transporter를 사용한다면 생산 공정을 간소화하고 비용을 줄일 수는 있지만 repebody는 양전하를 띠어 분비 효율이 낮다. 따라서 등전점(pI)이 높은 repebody의 등전점을 낮추어 음전하를 띄도록 해야 한다. 이것을 위해 repebody의 N 말단과 C 말단에 연속된 아스파탐산을 붙여 보았지만 분비가 증가하지 않았다. 다른 방법으로 ABC transporter를 통한 repebody 분비 효율을 높이기 위해 repebody의 항원 결합 부위의 반대쪽에 존재하는 열다섯 개의 양전하 아미노산을 아스파탐산으로 변환하여 repebody 표면이 강한 음전하를 띠도록 하였다. 그 결과, 기존 repebody의 발현 단백질 당 분비효율은 21.2%였으나 변형한 과음전하 repebody의 분비효율은 58.5%로 향상되었다. 결론적으로 과음전하를 통해 만들어진 repebody는 P. fluorescens에 의해 세포 바깥에 분비 생산할 수 있었다. A repebody, an artificial non-immunoglobulin protein scaffold, is expected to be a solution in the search for faster, cheaper, and customizable antibodies. However, the production of medical repebodies remains difficult due to their low yield and the complex purification processes required. The Pseudomonas fluorescens ABC transporter system has been suggested as an efficient and cost-effective method for repebody production, but the total yield is low because of the secreted protein’s positive charge; thus, a repebody with a high isoelectric point needs to be changed into a more negatively charged protein for better secretion. To achieve this, we first attached oligo-aspartic acids to the N- and C-terminals of the repebody, but secretion efficiency was not enhanced significantly. Subsequently, we devised an alternative method for improved secretion efficiency by engineering fifteen positively charged amino acids to aspartic acid in the non-antigen binding sites of the repebody to give a high net negative charge. As a result, secretion efficiency was greatly enhanced from 21.2% (wildtype) to 58.5% (negatively supercharged). The negatively supercharged repebody was succussfully produced extracellularly by ABC transporter secretion system in P. fluorescens.