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A chemical biology route to site-specific authentic protein modifications
Yang, Aerin,Ha, Sura,Ahn, Jihye,Kim, Rira,Kim, Sungyoon,Lee, Younghoon,Kim, Jaehoon,Sö,ll, Dieter,Lee, Hee-Yoon,Park, Hee-Sung American Association for the Advancement of Scienc 2016 Science Vol.354 No.6312
<P>Many essential biological processes are controlled by posttranslational protein modifications. The inability to synthetically attain the diversity enabled by these modifications limits functional studies of many proteins. We designed a three-step approach for installing authentic posttranslational modifications in recombinant proteins. We first use the established O-phosphoserine (Sep) orthogonal translation system to create a Sep-containing recombinant protein. The Sep residue is then dephosphorylated to dehydroalanine (Dha). Last, conjugate addition of alkyl iodides to Dha, promoted by zinc and copper, enables chemoselective carbon-carbon bond formation. To validate our approach, we produced histone H3, ubiquitin, and green fluorescent protein variants with site-specific modifications, including different methylations of H3K79. The methylated histones stimulate transcription through histone acetylation. This approach offers a powerful tool to engineer diverse designer proteins.</P>
Chemical biology approaches for studying posttranslational modifications
Yang, Aerin,Cho, Kyukwang,Park, Hee-Sung Informa UK (TaylorFrancis) 2018 RNA BIOLOGY Vol.15 No.4
<P>Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.</P>
Yang, Wonjun,Yoon, Aerin,Lee, Sanghoon,Kim, Soohyun,Han, Jungwon,Chung, Junho Nature Publishing Group 2017 Experimental and molecular medicine Vol.49 No.3
<P>Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the <I>Escherichia coli</I> pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones.</P>
Lee, Sangsik,Oh, Seunghee,Yang, Aerin,Kim, Jihyo,Sö,ll, Dieter,Lee, Daeyoup,Park, Hee‐,Sung WILEY‐VCH Verlag 2013 Angewandte Chemie Vol.125 No.22
<P><I><B>Einen selektiven Phosphoserin‐Einbau</B></I> beschreiben H.‐S. Park et al. in ihrer Zuschrift S. 5883 ff. Eine allgemeine Strategie für den Aufbau rekombinanter Histone mit ortsspezifischer Serinphosphorylierung wurde entwickelt, die auf der Modifizierung einer Phosphoseryl‐tRNA‐Synthetase (SepRS) und des Elongationsfaktors Tu (EF‐Tu) beruht. Die Methode dürfte die Erforschung der Histonphosphorylierung und kreuzregulatorischer Mechanismen vereinfachen.</P>
한정원,이종혁,윤수민,Aerin Yoon,황도빈,Hwa K Lee,Min S Kim,이유진,Won J Yang,박선영,윤홍덕,김효리,정준호 생화학분자생물학회 2016 Experimental and molecular medicine Vol.48 No.-
The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.
신생아의 출생 체중에 따른 혈액 여과지 17alpha-hydroxyprogesterone의 농도 분석 및 판정 기준 조정
박승만,권애린,양송현,박은아,최재황,황미정,남현경,이은희,Park, Seungman,Kwon, Aerin,Yang, Songhyeon,Park, Euna,Choi, Jaehwang,Hwang, Mijung,Nam, Hyeongyeong,Lee, Eunhee 대한유전성대사질환학회 2014 대한유전성대사질환학회지 Vol.14 No.2
The measurement of $17{\alpha}$-hydroxyprogesterone ($17{\alpha}$-OHP) in a dried blood spot on filter paper is an important for screening of congenital adrenal hyperplasia (CAH). Since high levels of $17{\alpha}$-OHP are frequently observed in premature infants without congenital adrenal hyperplasia, we evaluated cuts-off based on birth weight and performed validation. Birth weight and $17{\alpha}$-OHP concentration data of 292,204 newborn screening subjects in Greencross labopratories were analyzed. The cut-off values based on birth weight were newly evaluated and validated with the original data. The mean $17{\alpha}$-OHP concentration were 7.25 ng/mL in very low birth weight (VLBW) group, 4.02 ng/mL in low birth weight (LBW) group, 2.53 g/mL in normal birth weight (NBW) group, and 2.24 ng/mL in heavy birth weight (HBW) group. The cut-offs for CAH were decided as follows: 21.12 ng/mL for VLBW and LBW groups and 11.14 ng/mL for NBW and HBW groups. When applied new cut-offs for original data, positive rates in VLBW and LBW groups were decreased and positive rates in NBW and HBW groups were increased. The cut-offs based on birth weight should be used in the screening for CAH. We believe that our new cut-off reduce the false positive rate and false negative rate and our experience for cut-off set up and validation will be helpful for other laboratories doing newborn screening test.
Kim, Jihyo,Seo, Moon-Hyeong,Lee, Sangsik,Cho, Kyukwang,Yang, Aerin,Woo, Kyunghwa,Kim, Hak-Sung,Park, Hee-Sung American Chemical Society 2013 ANALYTICAL CHEMISTRY - Vol.85 No.3
<P>Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (<I>p</I>-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA<SUB>UCA</SUB> and <I>p</I>-acetylphenylalanyl-tRNA synthetase/tRNA<SUB>CUA</SUB>. The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2013/ancham.2013.85.issue-3/ac303089v/production/images/medium/ac-2012-03089v_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac303089v'>ACS Electronic Supporting Info</A></P>