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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

Gene expression of GFP in transgenic rice suspension cell cultures.A, Detection of GFP mRNA in non-transformed (NT) and selected transgenic rice suspension-cultured cells, including αAmy3sp-2, -5, and -9; CIN1sp-3, -5, and -9; and 33KDsp-1, -5, and -9. Total RNAs were isolated from NT and transgenic rice suspension-cultured cells, and then subjected to RT-PCR with specific GFP primers. The rice Act1 mRNA was used as an internal control. B, The proportion of medium GFP protein in various transgenic suspension cell lines. Total soluble proteins were extracted from each cell line, with a cell volume of 0.5 mL, in 1 mL of extraction buffer. A total of 1 mL of culture medium for each line was collected. Twenty microliters of total cellular protein (C) and 20 μL of total extracellular protein (M) were subjected to Western blot analysis using GFP antibodies. GFP proteins within cells and medium were detected, and compared with the total amount of GFP protein. The proportion of medium GFP for each independent cell line is presented as the total GFP in 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.g004: Gene expression of GFP in transgenic rice suspension cell cultures.A, Detection of GFP mRNA in non-transformed (NT) and selected transgenic rice suspension-cultured cells, including αAmy3sp-2, -5, and -9; CIN1sp-3, -5, and -9; and 33KDsp-1, -5, and -9. Total RNAs were isolated from NT and transgenic rice suspension-cultured cells, and then subjected to RT-PCR with specific GFP primers. The rice Act1 mRNA was used as an internal control. B, The proportion of medium GFP protein in various transgenic suspension cell lines. Total soluble proteins were extracted from each cell line, with a cell volume of 0.5 mL, in 1 mL of extraction buffer. A total of 1 mL of culture medium for each line was collected. Twenty microliters of total cellular protein (C) and 20 μL of total extracellular protein (M) were subjected to Western blot analysis using GFP antibodies. GFP proteins within cells and medium were detected, and compared with the total amount of GFP protein. The proportion of medium GFP for each independent cell line is presented as the total GFP in 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: To evaluate the secretion efficiency of the three signal peptides further, three independent suspension-cultured cell lines for each signal peptide were established. The mRNA levels of GFP in the various suspension cell lines were similar, except for line 2 of αAmy3sp-GFP, which had a slightly lower level, and line 3 of CIN1sp-GFP, which had a higher level than the other cell lines (Fig 4A). Each selected rice suspension-cultured cell line was cultured in MS medium for 5, 7, and 9 days, after which total cellular soluble protein and culture medium protein were obtained. The relative levels of GFP was detected by Western blot analysis with GFP antibodies, and quantified using Gel-Pro Analyzer (Fig 4B). The GFP proteins were detected in both intracellular and medium total soluble proteins of most suspension cell lines (Fig 4B). Compared with the αAmy3sp-GFP suspension cell lines and the CIN1sp-GFP suspension cell lines, the 33KDsp-GFP suspension cell lines accumulated more recombinant GFP proteins in the culture medium, regardless of the duration of the culture period (Fig 4B).


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)

Gene expression of GFP in transgenic rice suspension cell cultures.A, Detection of GFP mRNA in non-transformed (NT) and selected transgenic rice suspension-cultured cells, including αAmy3sp-2, -5, and -9; CIN1sp-3, -5, and -9; and 33KDsp-1, -5, and -9. Total RNAs were isolated from NT and transgenic rice suspension-cultured cells, and then subjected to RT-PCR with specific GFP primers. The rice Act1 mRNA was used as an internal control. B, The proportion of medium GFP protein in various transgenic suspension cell lines. Total soluble proteins were extracted from each cell line, with a cell volume of 0.5 mL, in 1 mL of extraction buffer. A total of 1 mL of culture medium for each line was collected. Twenty microliters of total cellular protein (C) and 20 μL of total extracellular protein (M) were subjected to Western blot analysis using GFP antibodies. GFP proteins within cells and medium were detected, and compared with the total amount of GFP protein. The proportion of medium GFP for each independent cell line is presented as the total GFP in 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.g004: Gene expression of GFP in transgenic rice suspension cell cultures.A, Detection of GFP mRNA in non-transformed (NT) and selected transgenic rice suspension-cultured cells, including αAmy3sp-2, -5, and -9; CIN1sp-3, -5, and -9; and 33KDsp-1, -5, and -9. Total RNAs were isolated from NT and transgenic rice suspension-cultured cells, and then subjected to RT-PCR with specific GFP primers. The rice Act1 mRNA was used as an internal control. B, The proportion of medium GFP protein in various transgenic suspension cell lines. Total soluble proteins were extracted from each cell line, with a cell volume of 0.5 mL, in 1 mL of extraction buffer. A total of 1 mL of culture medium for each line was collected. Twenty microliters of total cellular protein (C) and 20 μL of total extracellular protein (M) were subjected to Western blot analysis using GFP antibodies. GFP proteins within cells and medium were detected, and compared with the total amount of GFP protein. The proportion of medium GFP for each independent cell line is presented as the total GFP in 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: To evaluate the secretion efficiency of the three signal peptides further, three independent suspension-cultured cell lines for each signal peptide were established. The mRNA levels of GFP in the various suspension cell lines were similar, except for line 2 of αAmy3sp-GFP, which had a slightly lower level, and line 3 of CIN1sp-GFP, which had a higher level than the other cell lines (Fig 4A). Each selected rice suspension-cultured cell line was cultured in MS medium for 5, 7, and 9 days, after which total cellular soluble protein and culture medium protein were obtained. The relative levels of GFP was detected by Western blot analysis with GFP antibodies, and quantified using Gel-Pro Analyzer (Fig 4B). The GFP proteins were detected in both intracellular and medium total soluble proteins of most suspension cell lines (Fig 4B). Compared with the αAmy3sp-GFP suspension cell lines and the CIN1sp-GFP suspension cell lines, the 33KDsp-GFP suspension cell lines accumulated more recombinant GFP proteins in the culture medium, regardless of the duration of the culture period (Fig 4B).

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