Structure of PAP-IgM FcK fusion protein with J-chain expressed in transgenic plant

Kang, Yangjoo; Kim, Deuk-Su; Kim, Ki-Bum; Myung, Soon‐Chul; Oh, Yoo Jin; Park, Sungsu; Hinterdorfer, Peter; Ko, Kisung

doi:10.2478/ebtj-2023-0006

Cited by 6 publications

(8 citation statements)

References 35 publications

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“…Recent studies on recombinant proteins that utilize the Fc region of IgM have shown that the majority of these proteins predominantly exist in a mixed state, comprised of both monomers and oligomers. [60][61][62] All subsequent experiments in this study were carried out using this mixed form of ACE2 μK without any further fractionation. ACE2 μK and ACE2 K were confirmed to bind with all five investigated SARS-CoV-2 RBD variants, although there were differences in the degree of binding.…”

Section: Discussionmentioning

confidence: 99%

Enhanced binding and inhibition of SARS‐CoV‐2 by a plant‐derived ACE2 protein containing a fused mu tailpiece

Lim,

Kwon,

Jeong

et al. 2023

Biotechnology Journal

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Infectious diseases such as Coronavirus disease 2019 (COVID‐19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID‐19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). The angiotensin‐converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS‐CoV‐2 infection. Inhibition of the interaction between SARS‐CoV‐2 and ACE2 has been proposed as a target for the prevention and treatment of COVID‐19. We produced four recombinant plant‐derived ACE2 isoforms with or without the mu tailpiece (μ‐tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant‐derived ACE2 isoforms bound whole SARS‐CoV‐2 virus and the isolated receptor binding domains of SARS‐CoV‐2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of μ‐tp and KDEL to the ACE2 protein (ACE2 μK) had enhanced binding activity with SARS‐CoV‐2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant‐derived ACE2 μK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS‐CoV‐2 infection. The efficient and rapid scalability of plant‐derived ACE2 μK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.This article is protected by copyright. All rights reserved

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Section: Discussionmentioning

confidence: 99%

Enhanced binding and inhibition of SARS‐CoV‐2 by a plant‐derived ACE2 protein containing a fused mu tailpiece

Lim,

Kwon,

Jeong

et al. 2023

Biotechnology Journal

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“…2022; Kang et al . 2023). A 1 μL volume of each immunized and non‐immunized hemolymph was mixed with 19 μL of 1× PBS and 4 μL of a loading buffer (5% 2‐mercaptoethanol, 50% glycerol, 100 mM Tris–HCl, 10% SDS and 0.1% bromophenol blue) and loaded onto a 12% SDS‐PAGE gel.…”

Section: Methodsmentioning

confidence: 99%

“…SDS-PAGE was conducted to confirm the induction of attacins in immunized pupae, as described in previous report (Ko et al 1999;Lim et al 2022;Kang et al 2023). A 1 μL volume of each immunized and non-immunized hemolymph was mixed with 19 μL of 1× PBS and 4 μL of a loading buffer (5% 2-mercaptoethanol, 50% glycerol, 100 mM Tris-HCl, 10% SDS and 0.1% bromophenol blue) and loaded onto a 12% SDS-PAGE gel.…”

Section: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (S...mentioning

confidence: 99%

Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7‐501 using ion‐exchange chromatography, hydrophobic interaction chromatography, and Rotofor® isoelectric focusing

Kim

et al. 2023

Entomological Research

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Hyalophora cecropia pupae were infected by Enterobacter cloacae C7‐501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Ion‐exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non‐specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7‐501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.

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“…Plants are considered a heterologous bio-system for the production of valuable recombinant immunotherapeutic antibody proteins as an alternative to mammalian cell-based systems 5 . In plant expression system, the recombinant antibody genes are controlled by the CaMV35S promoter, which is considered a consistent and non-tissue-specific promoter in plant leaf and is mainly used for its stable or transient expression 6 – 8 . The transgene expression by the cauliflower mosaic virus (CaMV) 35S promoter has been investigated in leaf, root, and stem tissues 9 .…”

Section: Backgrounds and Summarymentioning

confidence: 99%

Single-cell transcriptome sequencing of plant leaf expressing anti-HER2 VHH–FcK cancer therapeutic protein

Kim,

Lee,

Kim

et al. 2023

Sci Data

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The transgenic plant is a promising strategy for the production of highly valuable biotherapeutic proteins such as recombinant vaccines and antibodies. To achieve an efficient level of protein production, codon sequences and expression cassette elements need to be optimized. However, the systematical expression of recombinant proteins in plant biomass can generally be controlled for the production of therapeutic proteins after the generation of transgenic plants. Without understanding the transgene expression patterns in plant tissue, it is difficult to enhance further production levels. In this study, single-cell RNA-sequencing (scRNA-seq) analysis of transgenic tobacco (Nicotiana tabacum) leaf, expressing an immunotherapeutic llama antibody against breast cancer, anti-HER2 VHH–Fc, was conducted to obtain data on the expression pattern of tissue-specific cells. These high-quality scRNA-seq data enabled the identification of gene expression patterns by cell types, which can be applied to select the best cell types or tissues for the high production of these recombinant antibodies. These data provide a foundation to elucidate the mechanisms that regulate the biosynthesis of recombinant proteins in N. tabacum.

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Structure of PAP-IgM FcK fusion protein with J-chain expressed in transgenic plant

Cited by 6 publications

References 35 publications

Enhanced binding and inhibition of SARS‐CoV‐2 by a plant‐derived ACE2 protein containing a fused mu tailpiece

Enhanced binding and inhibition of SARS‐CoV‐2 by a plant‐derived ACE2 protein containing a fused mu tailpiece

Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7‐501 using ion‐exchange chromatography, hydrophobic interaction chromatography, and Rotofor® isoelectric focusing

Single-cell transcriptome sequencing of plant leaf expressing anti-HER2 VHH–FcK cancer therapeutic protein

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