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Nonviral production of human interleukin-7 in spodoptera frugiperda insect cells as a soluble recombinant protein.

Mirzaei M, Xu Y, Elias CB, Prakash S - J. Biomed. Biotechnol. (2008)

Bottom Line: This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins.Here we report plasmid construction, transfection, and consequent expression of hIL-7 using this nonlytic insect cell expression system.The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 3775 University Street, Montreal, PQ, Canada H3A 2B4.

ABSTRACT
Human interleukin-7 (hIL-7) is a cytokine secreted by the stromal cells of the red marrow. It is important for proliferation during certain stages of B-cell maturation and for T and NK cell survival, development, and homeostasis. It is a critical growth factor for enhancement and recovery of the immune T-cell. Because of its strong immunomodulatory effects, hIL-7 may become a valuable supplementary agent for immunotherapeutical treatments in patients with HIV infection or immunodeficiency. Human IL-7 has previously been produced in various protein expression systems. In this paper, we present an alternative expression system, in Spodoptera frugiperda cells, for the production of hIL-7 using nonlytic vector systems. This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins. Here we report plasmid construction, transfection, and consequent expression of hIL-7 using this nonlytic insect cell expression system. The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

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Related in: MedlinePlus

Growth curves of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a) Viable cell density, total cell density, and viability of stably transfected Sf9 with pIE1/153A (V4)/hIL-7 plasmid. Cells were seeded at a density of 5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and  viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).
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fig3: Growth curves of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a) Viable cell density, total cell density, and viability of stably transfected Sf9 with pIE1/153A (V4)/hIL-7 plasmid. Cells were seeded at a density of 5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).

Mentions: Stable expression was verified bymaintaining the Sf9hIL-7A1 polyclone in puromycin-free medium after theselection. The expression of hIL-7 remained stable during more than 15 cellpassages and cells could be maintained further in selection-free media withoutany decrease in their production. The celllines were successfully frozen and retrieved from liquid nitrogen using thesame procedures used for Sf9 cells. Figure 3 shows a growth curve over an 11-day period inSf900 II for Sf9hIL-7A1 compared to nontransfected Sf9 insect cells. The cells appeared healthy and displayed an averagedoubling time of 24 hours. As shown in Figure 3(a), cell densities increasedexponentially over the first six days and then appeared to plateau between dayssix to eight, reaching maximum densities of approximately 8 × 106 cells·mL−1. Culture viabilities remained high (above 95%) until dayseven but then dropped to below 70% by day eleven. Culture viabilitiesranged from 95% to 99.5%, with most determinations above 97% during theexponential stage, which was similar to nontransfected Sf9 insect cells (Figure3(b)).


Nonviral production of human interleukin-7 in spodoptera frugiperda insect cells as a soluble recombinant protein.

Mirzaei M, Xu Y, Elias CB, Prakash S - J. Biomed. Biotechnol. (2008)

Growth curves of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a) Viable cell density, total cell density, and viability of stably transfected Sf9 with pIE1/153A (V4)/hIL-7 plasmid. Cells were seeded at a density of 5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and  viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2568039&req=5

fig3: Growth curves of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a) Viable cell density, total cell density, and viability of stably transfected Sf9 with pIE1/153A (V4)/hIL-7 plasmid. Cells were seeded at a density of 5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).
Mentions: Stable expression was verified bymaintaining the Sf9hIL-7A1 polyclone in puromycin-free medium after theselection. The expression of hIL-7 remained stable during more than 15 cellpassages and cells could be maintained further in selection-free media withoutany decrease in their production. The celllines were successfully frozen and retrieved from liquid nitrogen using thesame procedures used for Sf9 cells. Figure 3 shows a growth curve over an 11-day period inSf900 II for Sf9hIL-7A1 compared to nontransfected Sf9 insect cells. The cells appeared healthy and displayed an averagedoubling time of 24 hours. As shown in Figure 3(a), cell densities increasedexponentially over the first six days and then appeared to plateau between dayssix to eight, reaching maximum densities of approximately 8 × 106 cells·mL−1. Culture viabilities remained high (above 95%) until dayseven but then dropped to below 70% by day eleven. Culture viabilitiesranged from 95% to 99.5%, with most determinations above 97% during theexponential stage, which was similar to nontransfected Sf9 insect cells (Figure3(b)).

Bottom Line: This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins.Here we report plasmid construction, transfection, and consequent expression of hIL-7 using this nonlytic insect cell expression system.The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 3775 University Street, Montreal, PQ, Canada H3A 2B4.

ABSTRACT
Human interleukin-7 (hIL-7) is a cytokine secreted by the stromal cells of the red marrow. It is important for proliferation during certain stages of B-cell maturation and for T and NK cell survival, development, and homeostasis. It is a critical growth factor for enhancement and recovery of the immune T-cell. Because of its strong immunomodulatory effects, hIL-7 may become a valuable supplementary agent for immunotherapeutical treatments in patients with HIV infection or immunodeficiency. Human IL-7 has previously been produced in various protein expression systems. In this paper, we present an alternative expression system, in Spodoptera frugiperda cells, for the production of hIL-7 using nonlytic vector systems. This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins. Here we report plasmid construction, transfection, and consequent expression of hIL-7 using this nonlytic insect cell expression system. The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

Show MeSH
Related in: MedlinePlus