Fe engineering of anti-virus therapeutic human lgGl to alter binding to the neonatal Fe receptor in plant

Yangjoo Kang,Salah Park,llchan Song,Dalmuri Han,HyeKyung Lee,HyunJoo An,Kisung Ko 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.07

Effect of Nitrogen Deficiency on Recombinant Protein Production and Dimerization and Growth in Transgenic Plants

Kisung Ko,Yangjoo Kang,Yong Kyoo Shin,Sang-Won Park 한국원예학회 2017 한국원예학회 학술발표요지 Vol.2017 No.10

Transient plant expression of Angiotensin converting enzyme 2 (ACE2)-Fc fused KDEL ER retention signal in Nicotiana benthamiana

Hyunjoo Hwang,Yangjoo Kang,Kibum Kim,Kisung Ko 한국당과학회 2022 한국당과학회 학술대회 Vol.2022 No.07

Acute respiratory syndrome coronavirus SARS-CoV-2 causing COVID-19 has been rapidly spreading worldwide since it occurred in December 2019. SARS-CoV-2 infection occurs as the receptor-binding domain (RBD) of the coronavirus envelope spike protein binds to the angiotensin-converting enzyme 2 (ACE2) receptor. ACE2 is a cell membrane protein activated when it is cleaved and released from the cell surface. The soluble ACE2 can act as a decoy receptor to neutralize the SARS-CoV-2 virus, which is a potential therapy for COVID-19. In this study, the recombinant ACE2 protein was produced in a transient Nicotiana benthamiana plant expression system. The ACE2 was cloned to a fragment crystallizable (Fc) tagged with the KDEL sequence, endoplasmic reticulum (ER) retention signal (ACE2-FcK) in pEAQ-HT, the Cowpea mosaic virus (CPMV)-based transient plant expression vector. To design the recombinant protein structure of ACE2-FcK, the transmembrane region of ACE2 was removed using a transmembrane region prediction program. Four N-glycosylation sites recognized were identified using the N-glycosylation prediction program. Finally, Agrobacterium (LBA4404) carrying pEAQ-HT ACE2-FcK vector was applied for transient expression of ACE2-FcK in N. benthamiana. The proteins (100kDa) were successfully expressed and purified from leaves using protein A affinity chromatography. Additionally, suggested that the plant-derived ACE2-FcK has bioactivities as a therapeutic agent for coronavirus. Taken together, the ACE2-FcK protein can be transiently produced with an optimized expression time and leaf position post-infiltration in plant.

Expression and function of plant-derived dual monoclonal antibodies for multiple cancers

CaiQuan JIN,YangJoo Kang,Kibum Kim,Kisung Ko 한국당과학회 2022 한국당과학회 학술대회 Vol.2022 No.07

Recombinant antibody proteins can be applied to treat many various cancers. They are primarily produced using mammalian, insect, and bacteria cell culture systems. However, plant expression systems have been currently developed to produce antibodies. In particular, production of recombinant therapeutic antibodies using plant expression systems have several advantages, which are lack of human pathogenic agents, efficient production cost, and easy large scale up. In this study, we generated transgenic Nicotiana tabacum plant expressing both anti-colorectal cancer large single chain (LSC) CO17-1AK and anti-human epidermal growth factor receptor 2 (HER2) VHH-FcK monoclonal antibodies (mAbs) by cross-pollinating with plants expressing LSC CO17-1AK and anti-HER2 VHH-FcK, respectively. Plants expressing both LSC CO17-1AK and anti-HER2 VHH-FcK were screened with polymerase chain reaction and Western blot analyses. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding of the LSC CO17-1AK and anti-HER2 VHH-FcK to the HER2 and the GA733 proteins, respectively. The result of this study indicated that both mAb LSC CO17-1AK and anti-HER2 VHH-FcK produced in plant have their specific binding activities against HER2 and the GA733 antigens similar to their parental antibodies, respectively.

Effect of IgG Fc-fusion and KDEL-ER retention signal on prostate-specific antigen expression in plant and its immune in mice

Shin Chunha,김기범,Kang Yangjoo,Kim Deuk-Su,Seo Young-Jin,박세라,김미경,Lee Young Koung,김도선,Ko Kisung 한국식물생명공학회 2022 Plant biotechnology reports Vol.16 No.6

Prostate-specifc antigen (PSA) is a protein highly expressed in cancer cells of the prostate gland. In this study, the PSA recombinant protein was fused to the human immunoglobulin G crystallizable fragment (Fc) with the endoplasmic reticulum (ER) Lys-Asp-Glu-Leu (KDEL) retention signal tag for the plant expression system. Agrobacterium-mediated plant transformation was applied to generate transgenic tobacco plants expressing PSA tagged to KDEL (PSAK), PSA-Fc, and PSA-Fc fused to KDEL (PSA-FcK). Western blot analysis showed that the PSA-FcK protein was the most highly expressed among the three diferent proteins in the transgenic plant leaf. The PSAK, PSA-Fc, and PSA- FcK proteins were successfully purifed from plant leaves. Reduced and non-reduced sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that the PSA proteins fused to Fc and FcK were dimerized, whereas the PSA itself was not dimerized. N-glycan analysis demonstrated that the N-glycan profle of the PSA-Fc was mainly the structure with α(1,3)-fucose (Fuc) or β(1,2)-xylose (Xyl) glycan residues, whereas the PSA-Fc tagged with the KDEL ER retention signal had mainly an oligomannose-type structure. ELISA and SPR showed that PSA-FcK had a higher binding activity to Fc gamma receptor I than PSA-Fc and human Fc. In addition, PSA-FcK induced anti-PSA, and thus has the potential to be utilized as a prostate cancer vaccine

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 Kibum,Kim Do Sun,Kim Moon-Soo,Ko Kisung 한국응용약물학회 2022 Biomolecules & Therapeutics(구 응용약물학회지) Vol.30 No.6

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

Transient Plant Expression of Recombinant Rabies Virus Glycoprotein: A strategy for Vaccine Production

Lee Yeji,Park Jeanho,Kim Yerin,Hwang Hyunjoo,Jin Caiquan,Oh Yoojin,Kang Yangjoo,Lee Daehwan,Song Minhyeok,Lee Yoonji,고기성,Hong Mineui 한국원예학회 2024 원예과학기술지 Vol.42 No.3

The rabies virus causes neurological infections, resulting in the deaths of over 60,000 people worldwide each year. Currently available rabies vaccines are attenuated virus vaccines, which carry the risk of transmission among both humans and animals. An exciting alternative to these traditional vaccines is the use of recombinant plant-based vaccines. In this study, we applied the rabies virus glycoprotein (RVGP) as a recombinant vaccine to be transiently expressed in plants. To achieve this, we fused human immunoglobulin G Fc with the KDEL sequence, a motif for endoplasmic reticulum (ER) retention (FcK), to the RVGP, to generate RVGP-FcK. We then cloned the RVGPFcK gene expression cassette into the pEAQ vector and agro-infiltrated Agrobacterium tumefaciens (LBA4404) carrying the pEAQ RVGP-FcK vector into the leaves of Nicotiana benthamiana for transient expression. A RT-PCR analysis confirmed the transcription of the RVGP-FcK gene, which was evident as early as four days post-infiltration (dpi). To optimize the spatial and temporal aspects of RVGP-FcK production, we conducted analyses of its expression levels at different leaf positions (top, middle, and base) and dpi. A western blot analysis demonstrated that the RVGP-FcK protein reached its highest expression level at 7 dpi in the top leaf position and at 5 dpi in the middle leaf position. However, no detectable expression was observed in the bottom leaves at any time point. Subsequently, we validated the functionality of RVGP-FcK through an ELISA analysis. The results revealed that RVGP-FcK was expressed and assembled into its functional form most effectively at 5 and 7 dpi in the top leaf position and at 5 and 7 dpi in the middle leaf position. Our findings demonstrate that the transient expression of a functional RVGP-FcK protein can be optimized spatially and temporally in plants.