Expression of Recombinant Human Octamer-Binding Transcription Factor 4 in Rice Suspension Cells

Huang, Lei; Sinaga, Desyanti Saulina; Tan, Chia-Chun; Hsieh, Shu-Ju Micky; Huang, Chi‐Hung

doi:10.3390/ijms22031409

Cited by 4 publications

(3 citation statements)

References 44 publications

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“…The αAmy3 signal peptide has been previously reported to effectively direct cargo proteins for secretion from cells (Chen et al 2004 ). Various recombinant proteins have been successfully secreted into the liquid culture media using the αAmy3 signal peptide (Hong et al 2006 ; Huang et al 2005 , 2021 , 2015 ; Liu et al 2015 ; Nam et al 2017 ; Sinaga et al 2021 ; Van Giap et al 2019 ). However, in this study, the endogenous αAmy3 signal peptide failed to direct the rhBMP2m recombinant protein from the selected rhBMP2m knock-in suspension cultures into the culture medium.…”

Section: Discussionmentioning

confidence: 99%

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Nguyen,

Wu,

Chang

et al. 2024

Appl Microbiol Biotechnol

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Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3’ splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 μg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter–signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins. Key points • The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2 • The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins • The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity

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Section: Discussionmentioning

confidence: 99%

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Nguyen,

Wu,

Chang

et al. 2024

Appl Microbiol Biotechnol

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“…For example, using this approach, various recombinant proteins such as acid alpha-glucosidase (GAA) at 37 mg/L [31], human serum albumin (HSA) at 76.4 mg/L [32], and CCP monoclonal antibody (CCP mAb) at 22 mg/L [17] were produced in a transgenic rice cell suspension culture. In another study, Huang et al [33] reported that using the RAmy3D promoter system in a rice suspension cell culture, recombinant human octamer-binding transcription factor 4 (Oct4) reached the highest yield of approximately 0.41% of total cellular soluble proteins after one day of induction. These exploits showcased the potential of transforming plants into bio-factories capable of large-scale production of recombinant proteins.…”

Section: Discussionmentioning

confidence: 99%

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture

Do,

Yang

2024

CIMB

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Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+—NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

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“…Protease activity within host cells can lead to the degradation of unnecessary proteins, including recombinant proteins ( 12 ). Among these proteases, cysteine proteases have been identified for their negative impact on recombinant proteins ( 13 ).…”

Section: Introductionmentioning

confidence: 99%

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania

Abdi Ghavidel,

Bandehpour,

Noori

et al. 2024

IJPA

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Background: Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L. Methods: we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo). Results: The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter. Conclusion: Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.

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Expression of Recombinant Human Octamer-Binding Transcription Factor 4 in Rice Suspension Cells

Cited by 4 publications

References 44 publications

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania

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