HEK293-6E Cell Line细胞株 -BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

HEK293-6E Cell Line细胞株

-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

Description

HEK 293-6E expression system enables the high yield production of viral vector and post-translationally modified recombinant proteins through suspension-growing, serum-free adapted cell lines. It provides an optimized, chemically defined formulation for serum-free suspension culture of this cell line. The cell line can be used in combination with the cumate and coumermycin switches, which enable producers to turn on the expression of a given protein only when needed. Shorter, functional forms of EBNA1 reduced the difficulty of obtaining stable clones, allowing the isolation and characterization of a new 293EBNA1 cell line, 293-6E, that stably expresses a truncated EBNA1 protein. Further enhancement of r-protein expression was achieved by co-expression of selected genes in cis or trans. Use of an expression cassette for transient co-expression of truncated forms of EBNA1 in cis or in trans increased r-protein production in 293EBNA1 and non-EBNA1 cells as well as in the new 293-6E cell line. A modified serum-free medium amended by the addition of various additives was shown to increase r-protein and viral vector production.

Benefits

• High yield, low cost protein production for research and biomanufacturing

• Transient or stable expression

• Family of validated pTT vectors

• Choice of proprietary cell lines or custom cell line development to best suit your expression needs

• Master cell bank documentation for commercial production

• Two switches (cumate and coumermycin) allow you to turn on and off the expression of a given gene during production

• Serum-free media enables easy recovery of recombinant proteins

• Compatible with commercially available feeds; custom fed batch development

Cell Culture

Cells were cultured in chemically defined F17 medium supplemented with 1 g/L pluronic F68, 4 mM L-glutamine and 25 mg/L G418. HEK293-6E cells were cultivated in 125 mL to 1 L polycarbonate shake flasksin a Minitron™ CO2 orbital shaker with 25 mm orbital at 37°C, 5% CO2 atmosphere and 110 rounds per minute (rpm) without exceeding 2×106 cells/mL during maintenance. Small cultures were performed in 24 to 6 well multiwell plates using a linear shaker placed in a CO2 incubator with humidified atmosphere at 150 rpm.

HEK293-6E Cell Preparation for Transfection

Purpose: The purpose of the guidelines below is to provide a procedure to generate HEK293-6E cells that allow optimal transient protein expression.

1. Materials, Conditions: Culture media: FreeStyle™ 293 Expression Medium (Invitrogen Cat. 12338-018) Incubator: 8% CO2, 37oC, humidified atmosphere. Orbital shaking 90 rpm for 1L culture, 130 rpm for smaller volumes. Labware: Ploycarbonate shake flasks with vented lids. The flasks can be re-used after washing and autoclaving, but have to be checked carefully for potential cracks to avoid media spills in the incubator. The following sizes are used for specific volumes:

2. Cell Numbers The following cell numbers guarantee optimal protein expression A. Grow cells at least until they are 2x10E6 but no denser than 3x10E6. B. When splitting the cells, dilute them to no less than 1x10E6. C. Cells need to double with a 24 hr doubling rate to be ready for transfection D. Ideally have the cells double two consecutive times at maximum doubling rate (“log phase”) before Transfection E. At transfections dilute cells to 1x10E6.