Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice

Lim, Chae-Yeon; Kim, Deuk-Su; Kang, Yangjoo; Lee, Ye Rin; Kim, Ki-Bum; Kim, Do Sun; Kim, Moon‐Soo; Ko, Kisung

doi:10.4062/biomolther.2022.103

Cited by 15 publications

(12 citation statements)

References 38 publications

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“…Purification of recombinant PAP-IgM FcK × J-chain Purification of recombinant PAP-IgM FcK × J-chain protein from transgenic plant leaves protein from transgenic plant leaves Recombinant PAP-IgM FcK × J-chain fusion protein was purified according to a previous study (Kim et al, 2021;Lim et al, 2022). Briefly, the homogenized plant leaves of extraction buffer were centrifuged at 8,800 ×g for 30 min at 4℃.…”

Section: Immunoblot Analysis Immunoblot Analysismentioning

confidence: 99%

“…After the saturated suspension was incubated for overnight at 4℃. The suspension was centrifuged at 8,800 ×g for 30 min at 4℃, the pellet was resuspended in 1× PBS to 1/10 of the previous volume, and the final suspension was then centrifuged at 10,200 ×g for 30 min at 4℃ (Kim et al, 2021;Lim et al, 2022). The supernatant was then filtered through a hydrophilic PVDF 0.45 μm filter (Millipore, Billerica, MA).…”

Section: Immunoblot Analysis Immunoblot Analysismentioning

confidence: 99%

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

Kang

Kim

et al. 2023

The EuroBiotech Journal

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Transgenic plants expressing immunoglobulin (Ig) M Fc-fused Prostate acid phosphatase (PAP) antigenic proteins (PAP-IgM FcK) and J-chain proteins were generated by Agrobacterium-mediated transformation. The Fc region was tagged with the ER retention motif (KDEL) to make PAP-IgM FcK. Two transgenic plants were crossed together to generate F1 expressing both PAP-IgM FcK and J-chain proteins (PAP-IgM FcK × J-chain). PCR and RT-PCR analyses confirmed the transgene insertion and mRNA transcription of PAP-IgM FcK and J-chain in leaf tissue of PAP-IgM FcK × J-chain F1 plant. Western blot confirmed the expression of PAP-IgM FcK × J-chain protein. Size exclusion (SEC)-high performance liquid chromatography (HPLC) and Bio-transmission electron microscope (TEM) analyses were performed to show the size and shape of the PAP- IgM FcK × J-chain fusion proteins. These results suggest that PAP-IgM FcK with J-chain can be produced in plant expression system with plant crossing.

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Section: Immunoblot Analysis Immunoblot Analysismentioning

confidence: 99%

Section: Immunoblot Analysis Immunoblot Analysismentioning

confidence: 99%

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

Kang

Kim

et al. 2023

The EuroBiotech Journal

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“…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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“…Immobilized metal-ion affinity chromatography is widely used for purification of hexahistidine tagged proteins ( Vafaee and Alizadeh, 2018 ; Islam et al., 2019 ; Hanittinan et al., 2020 ; Islam et al., 2020 ; Marques et al., 2020 ; Soni et al., 2022 ). Other techniques such as one-step cation-exchange chromatography, Protein G-/A-based affinity chromatography, diafiltration (antibody purification) and polyelectrolyte precipitation (removal of plant proteins), hydrophobic interaction chromatography (HIC) followed by hydrophobic charge induction chromatography (HCIC) are employed in recombinant plant protein purification ( Fulton et al., 2015 ; Park et al., 2015 ; Shi et al., 2019 ; Miura et al., 2020 ; Lim et al., 2022 ; Grandits et al., 2023 ).…”

Section: Systems Engineering Approaches To Produce Recombinant Biopha...mentioning

confidence: 99%

Artificial intelligence-driven systems engineering for next-generation plant-derived biopharmaceuticals

Parthiban,

Vijeesh,

Gayathri

et al. 2023

Front. Plant Sci.

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Recombinant biopharmaceuticals including antigens, antibodies, hormones, cytokines, single-chain variable fragments, and peptides have been used as vaccines, diagnostics and therapeutics. Plant molecular pharming is a robust platform that uses plants as an expression system to produce simple and complex recombinant biopharmaceuticals on a large scale. Plant system has several advantages over other host systems such as humanized expression, glycosylation, scalability, reduced risk of human or animal pathogenic contaminants, rapid and cost-effective production. Despite many advantages, the expression of recombinant proteins in plant system is hindered by some factors such as non-human post-translational modifications, protein misfolding, conformation changes and instability. Artificial intelligence (AI) plays a vital role in various fields of biotechnology and in the aspect of plant molecular pharming, a significant increase in yield and stability can be achieved with the intervention of AI-based multi-approach to overcome the hindrance factors. Current limitations of plant-based recombinant biopharmaceutical production can be circumvented with the aid of synthetic biology tools and AI algorithms in plant-based glycan engineering for protein folding, stability, viability, catalytic activity and organelle targeting. The AI models, including but not limited to, neural network, support vector machines, linear regression, Gaussian process and regressor ensemble, work by predicting the training and experimental data sets to design and validate the protein structures thereby optimizing properties such as thermostability, catalytic activity, antibody affinity, and protein folding. This review focuses on, integrating systems engineering approaches and AI-based machine learning and deep learning algorithms in protein engineering and host engineering to augment protein production in plant systems to meet the ever-expanding therapeutics market.

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Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice

Cited by 15 publications

References 38 publications

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

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

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

Artificial intelligence-driven systems engineering for next-generation plant-derived biopharmaceuticals

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