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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

Production of secretory recombinant mGM-CSF in rice cells using the 33KD signal peptide.A, Expression vector containing a 33KD signal peptide fused with mGM-CSF chimeric gene. The 33kDsp-mGM-CSF fusion gene was engineered to be located downstream of the maize ubiquitin promoter (UbiP). B, Detection of mGM-CSF mRNA in non-transformed (NT) and selected 33KDsp-mGMCSF putative transgenic rice calli. Total RNAs were isolated from NT and transgenic rice calli, and then subjected to RT-PCR with specific mGM-CSF primers. The rice Act1 mRNA was used as an internal control. C & D, The proportion of medium rice-derived mGM-CSF (rmGM-CSF) protein in transgenic suspension cell line 10. Suspension cells with cell volume of 0.5 mL were cultured in 1 mL of medium with (C) or without sugar (D). Total soluble proteins were isolated from the suspension cells in 1 mL of protein extraction buffer, and 1 mL of culture medium was collected at various time periods as indicated. Twenty microliters of total cellular proteins (C) and 20 μL of total extracellular proteins (M) were subjected to Western blot analysis using mGM-CSF antibodies. The signals for rmGM-CSF within cells and medium were detected and compared with the total amount of rmGM-CSF protein. The proportion of medium rmGM-CSF is presented as the total rmGM-CSF in the medium compared with the total rmGM-CSF. A dark gray bar indicates medium rmGM-CSF, while a light gray bar indicates cellular rmGM-CSF. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. E, Production of mGM-CSF in transgenic rice suspension cells cultured in sugar-containing medium. The amount of recombinant mGM-CSF produced was measured in culture medium and rice suspension cells at 5, 7, and 9 days. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. F, Analysis of the biological activity of secreted rmGM-CSF from rice suspension cell culture medium at day 9. A murine hematopoietic cell line (NFS-60) was incubated with 60 ng/mL rice-derived mGM-CSF (R60). Commercial mGM-CSF derived from E. coli cells (E60; 60 ng/mL) was used as a reference standard. Both sucrose-free MS medium (C) and non-transformed rice culture medium (N) were used as negative controls. The proliferation of NFS-60 cells was measured with the MTT assay system. The error bar represents the standard deviation from triplicate cultures.
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pone.0140812.g006: Production of secretory recombinant mGM-CSF in rice cells using the 33KD signal peptide.A, Expression vector containing a 33KD signal peptide fused with mGM-CSF chimeric gene. The 33kDsp-mGM-CSF fusion gene was engineered to be located downstream of the maize ubiquitin promoter (UbiP). B, Detection of mGM-CSF mRNA in non-transformed (NT) and selected 33KDsp-mGMCSF putative transgenic rice calli. Total RNAs were isolated from NT and transgenic rice calli, and then subjected to RT-PCR with specific mGM-CSF primers. The rice Act1 mRNA was used as an internal control. C & D, The proportion of medium rice-derived mGM-CSF (rmGM-CSF) protein in transgenic suspension cell line 10. Suspension cells with cell volume of 0.5 mL were cultured in 1 mL of medium with (C) or without sugar (D). Total soluble proteins were isolated from the suspension cells in 1 mL of protein extraction buffer, and 1 mL of culture medium was collected at various time periods as indicated. Twenty microliters of total cellular proteins (C) and 20 μL of total extracellular proteins (M) were subjected to Western blot analysis using mGM-CSF antibodies. The signals for rmGM-CSF within cells and medium were detected and compared with the total amount of rmGM-CSF protein. The proportion of medium rmGM-CSF is presented as the total rmGM-CSF in the medium compared with the total rmGM-CSF. A dark gray bar indicates medium rmGM-CSF, while a light gray bar indicates cellular rmGM-CSF. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. E, Production of mGM-CSF in transgenic rice suspension cells cultured in sugar-containing medium. The amount of recombinant mGM-CSF produced was measured in culture medium and rice suspension cells at 5, 7, and 9 days. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. F, Analysis of the biological activity of secreted rmGM-CSF from rice suspension cell culture medium at day 9. A murine hematopoietic cell line (NFS-60) was incubated with 60 ng/mL rice-derived mGM-CSF (R60). Commercial mGM-CSF derived from E. coli cells (E60; 60 ng/mL) was used as a reference standard. Both sucrose-free MS medium (C) and non-transformed rice culture medium (N) were used as negative controls. The proliferation of NFS-60 cells was measured with the MTT assay system. The error bar represents the standard deviation from triplicate cultures.

Mentions: To examine whether 33KDsp can be applied for the efficient secretion of other valuable recombinant proteins, mGM-CSF was fused downstream of 33KDsp and expression was driven by the Ubi promoter (Fig 6A). Through the Agrobacterium-mediated transformation, several transgenic cell lines were obtained, and line 10 with a high level of mGM-CSF mRNA was selected to establish the rice suspension-cultured cell line (Fig 6B). The suspension-cultured cells were incubated in either sugar-containing medium for 5, 7, and 9 days or sugar-free medium for 1, 2, and 3 days, and then subjected to rmGM-CSF monitoring of the total soluble intracellular and medium proteins. As shown in Fig 6C, rice-derived mGM-CSF (rmGM-CSF) was detectable in both cell extract and culture medium, and its level increased with time in sugar-containing medium (Fig 6C). The rmGM-CSF was predominantly present in culture medium, at around 76%–92% of total rmGM-CSF protein (Fig 6C). The yields of rmGM-CSF were 8.5, 24.4, and 31.7 mg/L in sugar-containing medium cultured for 5, 7, and 9 days, respectively (Fig 6E). When the rmGM-CSF production by suspension cell line 10 was monitored upon culture in sugar-free medium for 1, 2, and 3 days, the protein was predominantly detected in the sugar-free culture medium, and the percentage of rmGM-CSF that was secreted reached 90% of the total amount produced (Fig 6D).


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)

Production of secretory recombinant mGM-CSF in rice cells using the 33KD signal peptide.A, Expression vector containing a 33KD signal peptide fused with mGM-CSF chimeric gene. The 33kDsp-mGM-CSF fusion gene was engineered to be located downstream of the maize ubiquitin promoter (UbiP). B, Detection of mGM-CSF mRNA in non-transformed (NT) and selected 33KDsp-mGMCSF putative transgenic rice calli. Total RNAs were isolated from NT and transgenic rice calli, and then subjected to RT-PCR with specific mGM-CSF primers. The rice Act1 mRNA was used as an internal control. C & D, The proportion of medium rice-derived mGM-CSF (rmGM-CSF) protein in transgenic suspension cell line 10. Suspension cells with cell volume of 0.5 mL were cultured in 1 mL of medium with (C) or without sugar (D). Total soluble proteins were isolated from the suspension cells in 1 mL of protein extraction buffer, and 1 mL of culture medium was collected at various time periods as indicated. Twenty microliters of total cellular proteins (C) and 20 μL of total extracellular proteins (M) were subjected to Western blot analysis using mGM-CSF antibodies. The signals for rmGM-CSF within cells and medium were detected and compared with the total amount of rmGM-CSF protein. The proportion of medium rmGM-CSF is presented as the total rmGM-CSF in the medium compared with the total rmGM-CSF. A dark gray bar indicates medium rmGM-CSF, while a light gray bar indicates cellular rmGM-CSF. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. E, Production of mGM-CSF in transgenic rice suspension cells cultured in sugar-containing medium. The amount of recombinant mGM-CSF produced was measured in culture medium and rice suspension cells at 5, 7, and 9 days. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. F, Analysis of the biological activity of secreted rmGM-CSF from rice suspension cell culture medium at day 9. A murine hematopoietic cell line (NFS-60) was incubated with 60 ng/mL rice-derived mGM-CSF (R60). Commercial mGM-CSF derived from E. coli cells (E60; 60 ng/mL) was used as a reference standard. Both sucrose-free MS medium (C) and non-transformed rice culture medium (N) were used as negative controls. The proliferation of NFS-60 cells was measured with the MTT assay system. The error bar represents the standard deviation from triplicate cultures.
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Related In: Results  -  Collection

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pone.0140812.g006: Production of secretory recombinant mGM-CSF in rice cells using the 33KD signal peptide.A, Expression vector containing a 33KD signal peptide fused with mGM-CSF chimeric gene. The 33kDsp-mGM-CSF fusion gene was engineered to be located downstream of the maize ubiquitin promoter (UbiP). B, Detection of mGM-CSF mRNA in non-transformed (NT) and selected 33KDsp-mGMCSF putative transgenic rice calli. Total RNAs were isolated from NT and transgenic rice calli, and then subjected to RT-PCR with specific mGM-CSF primers. The rice Act1 mRNA was used as an internal control. C & D, The proportion of medium rice-derived mGM-CSF (rmGM-CSF) protein in transgenic suspension cell line 10. Suspension cells with cell volume of 0.5 mL were cultured in 1 mL of medium with (C) or without sugar (D). Total soluble proteins were isolated from the suspension cells in 1 mL of protein extraction buffer, and 1 mL of culture medium was collected at various time periods as indicated. Twenty microliters of total cellular proteins (C) and 20 μL of total extracellular proteins (M) were subjected to Western blot analysis using mGM-CSF antibodies. The signals for rmGM-CSF within cells and medium were detected and compared with the total amount of rmGM-CSF protein. The proportion of medium rmGM-CSF is presented as the total rmGM-CSF in the medium compared with the total rmGM-CSF. A dark gray bar indicates medium rmGM-CSF, while a light gray bar indicates cellular rmGM-CSF. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. E, Production of mGM-CSF in transgenic rice suspension cells cultured in sugar-containing medium. The amount of recombinant mGM-CSF produced was measured in culture medium and rice suspension cells at 5, 7, and 9 days. Error bars indicate the standard deviation of three Western blot replicates from two biological repeats. F, Analysis of the biological activity of secreted rmGM-CSF from rice suspension cell culture medium at day 9. A murine hematopoietic cell line (NFS-60) was incubated with 60 ng/mL rice-derived mGM-CSF (R60). Commercial mGM-CSF derived from E. coli cells (E60; 60 ng/mL) was used as a reference standard. Both sucrose-free MS medium (C) and non-transformed rice culture medium (N) were used as negative controls. The proliferation of NFS-60 cells was measured with the MTT assay system. The error bar represents the standard deviation from triplicate cultures.
Mentions: To examine whether 33KDsp can be applied for the efficient secretion of other valuable recombinant proteins, mGM-CSF was fused downstream of 33KDsp and expression was driven by the Ubi promoter (Fig 6A). Through the Agrobacterium-mediated transformation, several transgenic cell lines were obtained, and line 10 with a high level of mGM-CSF mRNA was selected to establish the rice suspension-cultured cell line (Fig 6B). The suspension-cultured cells were incubated in either sugar-containing medium for 5, 7, and 9 days or sugar-free medium for 1, 2, and 3 days, and then subjected to rmGM-CSF monitoring of the total soluble intracellular and medium proteins. As shown in Fig 6C, rice-derived mGM-CSF (rmGM-CSF) was detectable in both cell extract and culture medium, and its level increased with time in sugar-containing medium (Fig 6C). The rmGM-CSF was predominantly present in culture medium, at around 76%–92% of total rmGM-CSF protein (Fig 6C). The yields of rmGM-CSF were 8.5, 24.4, and 31.7 mg/L in sugar-containing medium cultured for 5, 7, and 9 days, respectively (Fig 6E). When the rmGM-CSF production by suspension cell line 10 was monitored upon culture in sugar-free medium for 1, 2, and 3 days, the protein was predominantly detected in the sugar-free culture medium, and the percentage of rmGM-CSF that was secreted reached 90% of the total amount produced (Fig 6D).

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