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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

Detection of GFP protein in sugar-starved transgenic rice suspension cell cultures.A, Rice suspension cells (cell volume of 0.5 mL) were cultured in 1 mL of sugar-free MS medium for 2 and 3 days. Total cellular soluble proteins were isolated in 1 mL of protein extraction buffer from each of the treated cell cultures. Twenty microliters of total cellular protein (C) and 20 μL of total medium protein (M) were subjected to Western blot analysis using GFP antibodies. B, The proportion of medium GFP protein in various transgenic suspension cell lines. The amount of GFP within cells and medium was detected, and converted to the total amount of GFP protein. The proportion of medium GFP in each independent line is presented as the total GFP in the medium compared with the total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates cellular GFP. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats.
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pone.0140812.g005: Detection of GFP protein in sugar-starved transgenic rice suspension cell cultures.A, Rice suspension cells (cell volume of 0.5 mL) were cultured in 1 mL of sugar-free MS medium for 2 and 3 days. Total cellular soluble proteins were isolated in 1 mL of protein extraction buffer from each of the treated cell cultures. Twenty microliters of total cellular protein (C) and 20 μL of total medium protein (M) were subjected to Western blot analysis using GFP antibodies. B, The proportion of medium GFP protein in various transgenic suspension cell lines. The amount of GFP within cells and medium was detected, and converted to the total amount of GFP protein. The proportion of medium GFP in each independent line is presented as the total GFP in the medium compared with the total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates cellular GFP. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats.

Mentions: A recombinant protein expression platform in rice suspension cells has been established using a rice α-amylase gene promoter, αAmy3p, which drives the production of recombinant protein in sugar-free medium [29]. To evaluate the secretion efficiency of the three signal peptides in sugar-free growth conditions, two suspension-cultured cell lines per signal peptide were incubated in sugar-free medium for 2 and 3 days. As shown in Fig 5, in 2-day-cultured medium, there was substantially more accumulation of recombinant GFP proteins for the 33KDsp-GFP lines than for the αAmy3sp-GFP lines. When suspension-cultured cells were starved of sugar for 3 days, GFP proteins further accumulated in the culture medium, and even more GFP was secreted by the 33KDsp-GFP cell lines than by αAmy3sp-GFP. These results indicate that 33KDsp has higher secretion efficiency than αAmy3sp, and can be applied in a well-developed recombinant protein production platform using sugar-starved rice suspension-cultured cells.


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)

Detection of GFP protein in sugar-starved transgenic rice suspension cell cultures.A, Rice suspension cells (cell volume of 0.5 mL) were cultured in 1 mL of sugar-free MS medium for 2 and 3 days. Total cellular soluble proteins were isolated in 1 mL of protein extraction buffer from each of the treated cell cultures. Twenty microliters of total cellular protein (C) and 20 μL of total medium protein (M) were subjected to Western blot analysis using GFP antibodies. B, The proportion of medium GFP protein in various transgenic suspension cell lines. The amount of GFP within cells and medium was detected, and converted to the total amount of GFP protein. The proportion of medium GFP in each independent line is presented as the total GFP in the medium compared with the total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates cellular GFP. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608814&req=5

pone.0140812.g005: Detection of GFP protein in sugar-starved transgenic rice suspension cell cultures.A, Rice suspension cells (cell volume of 0.5 mL) were cultured in 1 mL of sugar-free MS medium for 2 and 3 days. Total cellular soluble proteins were isolated in 1 mL of protein extraction buffer from each of the treated cell cultures. Twenty microliters of total cellular protein (C) and 20 μL of total medium protein (M) were subjected to Western blot analysis using GFP antibodies. B, The proportion of medium GFP protein in various transgenic suspension cell lines. The amount of GFP within cells and medium was detected, and converted to the total amount of GFP protein. The proportion of medium GFP in each independent line is presented as the total GFP in the medium compared with the total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates cellular GFP. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats.
Mentions: A recombinant protein expression platform in rice suspension cells has been established using a rice α-amylase gene promoter, αAmy3p, which drives the production of recombinant protein in sugar-free medium [29]. To evaluate the secretion efficiency of the three signal peptides in sugar-free growth conditions, two suspension-cultured cell lines per signal peptide were incubated in sugar-free medium for 2 and 3 days. As shown in Fig 5, in 2-day-cultured medium, there was substantially more accumulation of recombinant GFP proteins for the 33KDsp-GFP lines than for the αAmy3sp-GFP lines. When suspension-cultured cells were starved of sugar for 3 days, GFP proteins further accumulated in the culture medium, and even more GFP was secreted by the 33KDsp-GFP cell lines than by αAmy3sp-GFP. These results indicate that 33KDsp has higher secretion efficiency than αAmy3sp, and can be applied in a well-developed recombinant protein production platform using sugar-starved rice suspension-cultured cells.

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