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Di-nanoperforator with Advanced and Broad-spectrum Anti-influenza Activity
Seokoh MOON,Hyunseok OH,Jeonghui MOON,YoungSeo PARK,Mi Soo KIM,Suhyun KIM,Dae-Hyuk KWEON 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
The sialyl receptor embedded loaded nanodiscs, aa discoidal lipid bilayer patch, inhibitsed influenza virus infection by erupting and entrapping viral RNPs in endolysosome. However, some structural improvements of nanodiscs were required for higher antiviral effects and wider targeting strains. Therefore, we applied intein mediated protein trans-splicing (PTS) for conjugating two mono-nanoperforators into di-nanoperforator. Each domain of intein was inserted to each terminal of membrane scaffold proteins which are major components of nanoperforator. The intein domain inserted membrane scaffold proteins were expressed and purified well for assembly of each nanoperforator. The PTS between two intein domain inserted mono-nanoperforators was proceeded well and di-nanoperforators as products show doubled size and molecular weight of mono-nanoperforators. The single receptor embedded homo di-nanoperforators reveal much higher antiviral effects against influenza virus than same concentrated mono-nanoperforators. Moreover, two different receptors (3’-sialic acid- and synthetic 6’-sialic acid-linked receptor) embedded hetero di-nanoperforator shows widen the targeting strains without interfering each antiviral effect unlikely to applying two receptors in mono-nanoperforators. Our results suggest doubling discs by Cfa intein-applied PTS advance virus-inhibiting action of nanodisc and potentials of numerous structural modifications in improving nanodisc technology.
Perforation of Membrane Envelope of Influenza A Virus by a Ganglioside-embedded Nanoparticle
Seokoh MOON,Byoungjae KONG,Yuna KIM,Younghun JUNG,Seok-Hyeon YU,Choongjin BAN,Jonghyeok SHIN,Joonbum PARK,Myungseo PARK,Chakhee KIM,Hooyeon KIM,Sora CHO,Yunjeong PARK,Hye Rin KIM,Hyun Seok OH,Jin Kyeo 한국생물공학회 2018 한국생물공학회 학술대회 Vol.2018 No.10
Moon, Seokoh,Kong, Byoungjae,Jung, Young-Hun,Kim, Yuna,Yu, Seokhyeon,Park, Joon-bum,Shin, Jonghyeok,Kweon, Dae-Hyuk Elsevier 2018 Process biochemistry Vol.66 No.-
<P><B>Abstract</B></P> <P>Membrane scaffold protein (MSP) is a versatile protein that can be used to study diverse membrane proteins. MSP is strongly expressed in <I>E. coli</I>; however, applications of MSP in <I>in vivo</I> studies remain limited because of contamination with large amounts of endotoxins. Endotoxins cannot be easily removed from MSP following standard purification protocols for His6-tagged proteins, washing with detergents, or Q-Sepharose anion exchange chromatography, regardless of whether the expression host is <I>E. coli</I> BL21(DE3) or ClearColi BL21(DE3). Furthermore, the concentrations of MSP-bound endotoxins were not reduced during nanodisc formation, such that the assembled nanodiscs still contained significant amounts of endotoxins. We hypothesized that the structural properties of MSP that are responsible for membrane scaffolding mediated the strong binding between MSP and the endotoxins. We showed that partial denaturation of MSP with 2M urea effectively disrupted MSP-endotoxin interactions. MSP-bound endotoxins were successfully removed <I>via</I> Q-Sepharose chromatography following urea treatment. The combined treatment with urea and Q-Sepharose resulted in ∼80-fold reduction in the specific endotoxin level relative to that of conventional Ni-NTA chromatography combined with detergent treatment. The low endotoxin level of 2.0EU/mg MSP obtained in this study makes it suitable for applications in animal studies.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Membrane scaffold protein is used to form nanodisc and has diverse biotechnological applications. </LI> <LI> MSPs purified by Ni-NTA chromatography have abnormally high levels of endotoxin. </LI> <LI> Conventional anion exchange chromatography was not effective for MSP purification. </LI> <LI> Endotoxins could be separated from MSP after partial unfolding of MSP by urea. </LI> <LI> The endotoxin-free MSPs and nanodiscs are suitable for <I>in vivo</I> studies. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>