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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 fluorescence in selected transgenic calli.A, Putative transgenic calli harboring the GFP gene were grown on hygromycin selection medium and observed by fluorescence microscopy. B, Rice calli (cell volume of 0.5 mL) were cultured in 1 mL of MS with sugar medium for 5, 7 and 9 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 silver staining. C, A standard curve for GFP protein amount and fluorescence intensity. D, The ratio of GFP protein in cellular extract and cultured medium among various transgenic cell lines. For each analyzed transgenic line, 0.5 mL rice calli were cultured in 1 mL of MS with sugar medium for 5 days. GFP fluorescence signals from the culture medium and total cellular soluble protein were measured. The upper panel indicated the amount of GFP protein, averaged by three replicated fluorescence detections from two biological repeats. C: amount of cellular GFP proteins (μg); M: total amount of GFP proteins in medium (μg); T: amount of total GFP proteins (μg). The standard deviation from each selected transgenic line was shown in S1 Table. The proportion of medium GFP in each independent line is presented as the GFP fluorescence from total GFP in medium compared with that from total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates intracellular GFP.
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pone.0140812.g003: Detection of GFP fluorescence in selected transgenic calli.A, Putative transgenic calli harboring the GFP gene were grown on hygromycin selection medium and observed by fluorescence microscopy. B, Rice calli (cell volume of 0.5 mL) were cultured in 1 mL of MS with sugar medium for 5, 7 and 9 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 silver staining. C, A standard curve for GFP protein amount and fluorescence intensity. D, The ratio of GFP protein in cellular extract and cultured medium among various transgenic cell lines. For each analyzed transgenic line, 0.5 mL rice calli were cultured in 1 mL of MS with sugar medium for 5 days. GFP fluorescence signals from the culture medium and total cellular soluble protein were measured. The upper panel indicated the amount of GFP protein, averaged by three replicated fluorescence detections from two biological repeats. C: amount of cellular GFP proteins (μg); M: total amount of GFP proteins in medium (μg); T: amount of total GFP proteins (μg). The standard deviation from each selected transgenic line was shown in S1 Table. The proportion of medium GFP in each independent line is presented as the GFP fluorescence from total GFP in medium compared with that from total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates intracellular GFP.

Mentions: To compare the efficacy of the three rice signal peptides for the efficient secretion of recombinant protein in rice cultured cells, and to reduce influence from other unexpected factors on the secretion efficiency of these signal peptides, ubiquitin (Ubi) promoter which drives constitutive gene expression both in sugar feeding and starving rice cultured cells was used to control the expression of reporter gene. Three chimeric genes, Ubi::αAmy3sp-GFP, Ubi::CIN1sp-GFP, and Ubi::33KDsp-GFP, containing αAmy3sp, CIN1sp, and 33KDsp, respectively, were generated (Fig 2). These expression cassettes were introduced individually into the rice genome via Agrobacterium-mediated transformation. Several transgenic lines were obtained for each signal peptide, and GFP fluorescence was observed as shown in Fig 3A. Nine transgenic lines of each construct were randomly selected to detect GFP levels. We previously demonstrated that the cell viability of suspension-cultured rice cells in sugar containing medium can be maintained 90% at least for 10 days [26]. We further confirmed the result by protein gel analysis, and the band patterns of intracellular soluble proteins were obviously different from cultured medium proteins at 5, 7 and 9 days (Fig 3B). Therefore, the transgenic calli were cultured in MS liquid medium for 5 days, and both cultured calli and medium were collected to prevent artifacts caused by cell lysis. To quantify the level of fluorescence of GFP, a standard curve was used with a concentration range from 0.1 to 3 μg/mL purified recombinant GFP protein (Fig 3C). For each independent cell line, the levels of recombinant GFP proteins in the culture medium and in cellular extracts were measured (Fig 3D; S1 Table), and the proportion of GFP protein secreted was determined by dividing the amount of GFP in the medium by the total GFP. As shown in Fig 3D, wide ranges of intracellular to medium GFP ratios were displayed among the selected lines. For αAmy3sp-GFP transgenic cell lines, 37%–95% of total GFP was detected in the medium. For CIN1spGFP transgenic cell lines, 0%–75% of total GFP was detected in the medium. However, for 33KDspGFP transgenic cell lines, 52%–95% of total GFP was detected in the medium, and in the majority of these selected lines, more than 80% of total GFP was present in the culture medium. This result showed that the highest proportion of medium GFP was achieved using 33KDspGFP-containing calli, followed by αAmy3spGFP and then CIN1spGFP, suggesting that the secretion efficiency of 33KDsp is higher than that of αAmy3sp and CIN1sp.


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 fluorescence in selected transgenic calli.A, Putative transgenic calli harboring the GFP gene were grown on hygromycin selection medium and observed by fluorescence microscopy. B, Rice calli (cell volume of 0.5 mL) were cultured in 1 mL of MS with sugar medium for 5, 7 and 9 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 silver staining. C, A standard curve for GFP protein amount and fluorescence intensity. D, The ratio of GFP protein in cellular extract and cultured medium among various transgenic cell lines. For each analyzed transgenic line, 0.5 mL rice calli were cultured in 1 mL of MS with sugar medium for 5 days. GFP fluorescence signals from the culture medium and total cellular soluble protein were measured. The upper panel indicated the amount of GFP protein, averaged by three replicated fluorescence detections from two biological repeats. C: amount of cellular GFP proteins (μg); M: total amount of GFP proteins in medium (μg); T: amount of total GFP proteins (μg). The standard deviation from each selected transgenic line was shown in S1 Table. The proportion of medium GFP in each independent line is presented as the GFP fluorescence from total GFP in medium compared with that from total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates intracellular GFP.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140812.g003: Detection of GFP fluorescence in selected transgenic calli.A, Putative transgenic calli harboring the GFP gene were grown on hygromycin selection medium and observed by fluorescence microscopy. B, Rice calli (cell volume of 0.5 mL) were cultured in 1 mL of MS with sugar medium for 5, 7 and 9 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 silver staining. C, A standard curve for GFP protein amount and fluorescence intensity. D, The ratio of GFP protein in cellular extract and cultured medium among various transgenic cell lines. For each analyzed transgenic line, 0.5 mL rice calli were cultured in 1 mL of MS with sugar medium for 5 days. GFP fluorescence signals from the culture medium and total cellular soluble protein were measured. The upper panel indicated the amount of GFP protein, averaged by three replicated fluorescence detections from two biological repeats. C: amount of cellular GFP proteins (μg); M: total amount of GFP proteins in medium (μg); T: amount of total GFP proteins (μg). The standard deviation from each selected transgenic line was shown in S1 Table. The proportion of medium GFP in each independent line is presented as the GFP fluorescence from total GFP in medium compared with that from total GFP. A dark gray bar indicates medium GFP, while a light gray bar indicates intracellular GFP.
Mentions: To compare the efficacy of the three rice signal peptides for the efficient secretion of recombinant protein in rice cultured cells, and to reduce influence from other unexpected factors on the secretion efficiency of these signal peptides, ubiquitin (Ubi) promoter which drives constitutive gene expression both in sugar feeding and starving rice cultured cells was used to control the expression of reporter gene. Three chimeric genes, Ubi::αAmy3sp-GFP, Ubi::CIN1sp-GFP, and Ubi::33KDsp-GFP, containing αAmy3sp, CIN1sp, and 33KDsp, respectively, were generated (Fig 2). These expression cassettes were introduced individually into the rice genome via Agrobacterium-mediated transformation. Several transgenic lines were obtained for each signal peptide, and GFP fluorescence was observed as shown in Fig 3A. Nine transgenic lines of each construct were randomly selected to detect GFP levels. We previously demonstrated that the cell viability of suspension-cultured rice cells in sugar containing medium can be maintained 90% at least for 10 days [26]. We further confirmed the result by protein gel analysis, and the band patterns of intracellular soluble proteins were obviously different from cultured medium proteins at 5, 7 and 9 days (Fig 3B). Therefore, the transgenic calli were cultured in MS liquid medium for 5 days, and both cultured calli and medium were collected to prevent artifacts caused by cell lysis. To quantify the level of fluorescence of GFP, a standard curve was used with a concentration range from 0.1 to 3 μg/mL purified recombinant GFP protein (Fig 3C). For each independent cell line, the levels of recombinant GFP proteins in the culture medium and in cellular extracts were measured (Fig 3D; S1 Table), and the proportion of GFP protein secreted was determined by dividing the amount of GFP in the medium by the total GFP. As shown in Fig 3D, wide ranges of intracellular to medium GFP ratios were displayed among the selected lines. For αAmy3sp-GFP transgenic cell lines, 37%–95% of total GFP was detected in the medium. For CIN1spGFP transgenic cell lines, 0%–75% of total GFP was detected in the medium. However, for 33KDspGFP transgenic cell lines, 52%–95% of total GFP was detected in the medium, and in the majority of these selected lines, more than 80% of total GFP was present in the culture medium. This result showed that the highest proportion of medium GFP was achieved using 33KDspGFP-containing calli, followed by αAmy3spGFP and then CIN1spGFP, suggesting that the secretion efficiency of 33KDsp is higher than that of αAmy3sp and CIN1sp.

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