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Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

Huang LF, Tan CC, Yeh JF, Liu HY, Liu YK, Ho SL, Lu CA - PLoS ONE (2015)

Bottom Line: The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.The extracellular yield of rmGM-CSF reached 31.7 mg/L.Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Taoyuan, Taiwan, ROC.

ABSTRACT
Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

No MeSH data available.


Related in: MedlinePlus

Expression vectors with signal peptides of αAmy3, OsCIN1, or Os33KD used for rice transformation.Various signal peptide cDNAs (αAmy3sp, CIN1sp, or 33KDsp) were fused in-frame upstream of green fluorescent protein (GFP) DNA. The fusion genes were engineered to be located downstream of the maize ubiquitin promoter (UbiP). β-glucuronidase (GUS) and the hygromycin-resistance gene (Hph) were both driven by a CaMV35S promoter (35SP). Constructs contained the left and right borders (LB, RB) flanking the T-DNA that can be transferred into the plant genome via Agrobacterium-mediated transformation. Arrows indicate specific primers used in this study. Amino acid sequences for αAmy3sp, CIN1sp, and 33KDsp are shown in the upper box. Carets indicate predicted signal peptide cleavage sites.
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pone.0140812.g002: Expression vectors with signal peptides of αAmy3, OsCIN1, or Os33KD used for rice transformation.Various signal peptide cDNAs (αAmy3sp, CIN1sp, or 33KDsp) were fused in-frame upstream of green fluorescent protein (GFP) DNA. The fusion genes were engineered to be located downstream of the maize ubiquitin promoter (UbiP). β-glucuronidase (GUS) and the hygromycin-resistance gene (Hph) were both driven by a CaMV35S promoter (35SP). Constructs contained the left and right borders (LB, RB) flanking the T-DNA that can be transferred into the plant genome via Agrobacterium-mediated transformation. Arrows indicate specific primers used in this study. Amino acid sequences for αAmy3sp, CIN1sp, and 33KDsp are shown in the upper box. Carets indicate predicted signal peptide cleavage sites.

Mentions: To identify a better signal peptide than rice αAmy3sp, secretory proteins naturally produced by rice suspension cells are good resources to test. The rice suspension cells were cultured in sugar-containing medium or sugar-free medium, for various periods, and the total protein in the medium was analyzed by SDS-PAGE and silver staining. A strong signal at a molecular weight of approximately 33 kDa was observed in both sugar-containing medium and sugar-free medium (Fig 1). The 33 kDa protein was purified by ammonium sulfate precipitation, and then followed by fast protein liquid chromatography. The protein sequence of the corresponding 33 kDa secretory protein was determined, and was identical to the DUF26-like protein/33KD secretory protein (called Os33KD and encoded by Os04g0659300), which has been shown as a secretory protein in the culture medium of rice suspension cells [47]. Thus, the signal peptide of Os33KD, called 33KDsp, was selected to compare the secretion efficiency of recombinant proteins with that of αAmy3sp. In addition to 33KDsp, another rice signal peptide was selected, a rice cell wall invertase (OsCIN1, encoded by Os02g0534400) which is present in the cell wall and hydrolyzes sucrose to glucose and fructose [48]; this signal peptide was named CIN1sp. The signal peptide regions of αAmy3, Os33KD, and OsCIN1 were predicted using SignalP 3.0 [49], which putatively showed that 25 amino acids at the N-terminus of αAmy3, 25 amino acids at the N-terminus of Os33KD, and 22 amino acids at the N-terminus of OsCIN1 are the signal peptides (Fig 2).


Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

Huang LF, Tan CC, Yeh JF, Liu HY, Liu YK, Ho SL, Lu CA - PLoS ONE (2015)

Expression vectors with signal peptides of αAmy3, OsCIN1, or Os33KD used for rice transformation.Various signal peptide cDNAs (αAmy3sp, CIN1sp, or 33KDsp) were fused in-frame upstream of green fluorescent protein (GFP) DNA. The fusion genes were engineered to be located downstream of the maize ubiquitin promoter (UbiP). β-glucuronidase (GUS) and the hygromycin-resistance gene (Hph) were both driven by a CaMV35S promoter (35SP). Constructs contained the left and right borders (LB, RB) flanking the T-DNA that can be transferred into the plant genome via Agrobacterium-mediated transformation. Arrows indicate specific primers used in this study. Amino acid sequences for αAmy3sp, CIN1sp, and 33KDsp are shown in the upper box. Carets indicate predicted signal peptide cleavage sites.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608814&req=5

pone.0140812.g002: Expression vectors with signal peptides of αAmy3, OsCIN1, or Os33KD used for rice transformation.Various signal peptide cDNAs (αAmy3sp, CIN1sp, or 33KDsp) were fused in-frame upstream of green fluorescent protein (GFP) DNA. The fusion genes were engineered to be located downstream of the maize ubiquitin promoter (UbiP). β-glucuronidase (GUS) and the hygromycin-resistance gene (Hph) were both driven by a CaMV35S promoter (35SP). Constructs contained the left and right borders (LB, RB) flanking the T-DNA that can be transferred into the plant genome via Agrobacterium-mediated transformation. Arrows indicate specific primers used in this study. Amino acid sequences for αAmy3sp, CIN1sp, and 33KDsp are shown in the upper box. Carets indicate predicted signal peptide cleavage sites.
Mentions: To identify a better signal peptide than rice αAmy3sp, secretory proteins naturally produced by rice suspension cells are good resources to test. The rice suspension cells were cultured in sugar-containing medium or sugar-free medium, for various periods, and the total protein in the medium was analyzed by SDS-PAGE and silver staining. A strong signal at a molecular weight of approximately 33 kDa was observed in both sugar-containing medium and sugar-free medium (Fig 1). The 33 kDa protein was purified by ammonium sulfate precipitation, and then followed by fast protein liquid chromatography. The protein sequence of the corresponding 33 kDa secretory protein was determined, and was identical to the DUF26-like protein/33KD secretory protein (called Os33KD and encoded by Os04g0659300), which has been shown as a secretory protein in the culture medium of rice suspension cells [47]. Thus, the signal peptide of Os33KD, called 33KDsp, was selected to compare the secretion efficiency of recombinant proteins with that of αAmy3sp. In addition to 33KDsp, another rice signal peptide was selected, a rice cell wall invertase (OsCIN1, encoded by Os02g0534400) which is present in the cell wall and hydrolyzes sucrose to glucose and fructose [48]; this signal peptide was named CIN1sp. The signal peptide regions of αAmy3, Os33KD, and OsCIN1 were predicted using SignalP 3.0 [49], which putatively showed that 25 amino acids at the N-terminus of αAmy3, 25 amino acids at the N-terminus of Os33KD, and 22 amino acids at the N-terminus of OsCIN1 are the signal peptides (Fig 2).

Bottom Line: The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.The extracellular yield of rmGM-CSF reached 31.7 mg/L.Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Taoyuan, Taiwan, ROC.

ABSTRACT
Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

No MeSH data available.


Related in: MedlinePlus