Editing Protein Expression 293 (section)

=== Discussion ===
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The 10-layer cell factories are an effective vessel for the production of milligram quantities of protein. A major advantage of using the cell factory over other traditional vessels, such as roller bottles, shake flasks or spinner flasks, is that they do not require the purchase of any additional laboratory equipment. A standard CO<sub>2</sub> incubator (~6.0 cu. ft.) will easily accommodate four 10-layer cell factories ('''Fig. 2'''). In addition, these vessels require less labor and space than dishes, flasks or roller bottles to produce cells and proteins; one 10-layer cell factory is equivalent to the use of 7.5 regular roller bottles ('''Table 1'''). More importantly, the HEK 293T cells adapt well in the vessels and are highly amenable to transient transfection by PEI, thus providing a low-cost and effective option to commercial transfection reagents. While cell factories are designed to be disposable cell culture plasticware, we have been able to efficiently clean and re-use these vessels multiple times without contamination issues, thus significantly reducing its cost per use. Transient protein expression using a 10-layer cell factory is capable of producing ~1-10 mg of purified protein ('''Fig. 4'''), depending on the protein and its modifications. It is possible to go from cloning and small-scale expression to completion of large-scale expression and purification in about 3-4 weeks. In summary, this is a rapid, general platform that is highly useful in the production of secreted human or viral surface proteins. Moreover, this platform can also be used to express antibodies, virus-like particles, and adenoviruses and lentiviruses for gene transfer.

We have listed below common problems and potential solutions For a more detailed troubleshooting guide, please see <sup>14</sup>.

#'''No or very low protein expression: '''
#*Poor health of the cells- Stain the cells using trypan blue for viability and test for mycoplasma contamination. If cells have a high passage number (>25 passages) and are growing slowly, start fresh with new HEK 293T cells. Transfection efficiency and protein production are affected by the age of the cells in culture.
#*Poor transient transfection- Cells should be transfected at a confluency level of 40%. The ratios of DNA to transfection reagent should be optimized.
#*Protein is unstable or not folded- Check the cell pellet for insoluble protein. Test the expression of other constructs or homologous protein.
#'''No protein is eluted from column:'''
#*Protein retained on the column- Elute the protein of interest with 0.1 M glycine pH 2.2 and analyze by western blot. If protein is retained, use a higher concentration of synthetic HA peptide for elution or incubate the column at 37 °C for longer periods.
#*anti-HA column is damaged- While the column, if cleaned and stored properly, can be reused multiple times, the efficiency of the column will decrease over time. Use a new anti-HA column.
#*Protein degradation- Add protease inhibitors and keep protein on ice during the entire purification process.
#*Protein has precipitated or aggregated on column- If the protein is unstable, try different constructs or buffer conditions (ie. pH, salts, additives, etc.) to improve protein stability.
#*Protein failed to bind to column- The HA-tag may be buried or inaccessible. Check to see that the protein is not in the flow through or wash.
#'''Serum albumin contamination:'''<br/>Bovine serum albumin (BSA) tends to bind non-specifically to the anti-HA resin and may contaminate the HA peptide elutions. In Fig. 4, BSA migrates as a compact band at an apparent molecular weight of ~60-65 kDa. In this case, increase the Tween-20 concentration in the 1X PBS-Tween20 wash to 0.2% (v/v) and increase the volume of the wash. Alternatively, BSA contamination may be removed using size exclusion or ion exchange chromatography.
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