Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

The overall goal of this mammalian protein expression technique is to produce milligram quantities of natively folded proteins suitable for structural, biophysical and functional studies. The main advantage of this technique is that it is a fast and straightforward protocol for obtaining, secreted mammalian proteins and a concentration in purity required for structural studies. Generally, we find that most issues with protein yield are due to cell health and viability.

By closely monitoring cell viability and also monitoring media sugar levels, you greatly improve both protein yield and prolong the usefulness of cells. It is also very important to monitor cell density. We find that if at any time before transfection the cells exceed 2 million cells per milliliter, then protein yield is greatly reduced To perform large scale culture of 2 93 F cells supplement one liter of 2 93 F media with 10 milliliters of 100 x glutamine and five milliliters of 100 x pen strep.

The antibiotic is at a sufficient strength in serum free conditions and the reduced antibiotic concentration improves cell viability during transfection, which improves protein yields culture. The 2 93 F cells in 300 milliliters of media in one liter, polycarbonate baffled erlenmeyer flasks with vented caps at 37 degrees Celsius with 8%carbon dioxide while shaking in a standard tissue culture incubator one day before transfection dilute the cells to 0.5 million cells per milliliter density on the day of transfection. Supplement the culture medium by adding 10%volume of 2%weight per volume cell boost in 2 93 F media.

Add kafu in seen at this step to control protein glycosylation. Prepare DNA and transfection reagent solutions in serum free media incubate for five minutes. Next, add the transfection reagent into the DNA solution in one milliliter increments mixing.

Gently incubate for 30 minutes at room temperature for reagent DNA complexes to form. Then add the solution onto the cells In a dropwise fashion, allow the transfected cells to express protein for 72 to 96 hours to purify the glycoprotein. First decant the culture into a centrifuge flask centrifuge for 20 minutes at 1300 Gs.To pellet the cells, collect the supernat and if necessary, spin a second time and or use 0.22 micron filter to clarify the supernatant.

Next, add 10%volume of 10 x nickel nitro load, tri acetic acid or nickel NTA binding buffer. Then prepare a gravity column at four degree Celsius by adding two milliliters of the nickel NTA slurry to a column and equilibrating with 10 column volumes of one x binding buffer working at four degrees Celsius. Flow the supernatant over the resin and to collect the flow through.

After pouring the supernatant flow through over the column wash with 10 column volumes of wash buffer. Then elute the protein in five column volumes of elution buffer. If deglycosylation is required for a final volume of 0.5 milliliters, concentrate EIT to 0.43 milliliters using a centrifugation concentrator pellet any debris by centrifugation at 16, 000 GS and four degrees Celsius.

Then add 50 microliters of 500 millimolar sodium citrate, pH 5.5 and 20 microliters of endo hf. Incubate the mixture at room temperature for two hours to remove the endo HF first wash amylose resin three times in phosphate buffered saline. Incubate the protein with the washed resin for one hour at four degrees Celsius.

Following incubation, spin for five minutes at 1000 Gs to pellet the resin and collect the supernatant. Concentrate the protein using an appropriate molecular weight cutoff, centrifugation filter, and buffer exchange into storage buffer shown. Here are representative SDS page results for a secreted protein expressed in cine treated cells following nickel affinity chromatography.

The first lane is the protein prior to deglycosylation, and the second lane is the protein following deglycosylation by endo hf. The glycosylated protein runs about 10 kilodaltons higher and then collapses to a single band consistent with the predicted molecular weight following deglycosylation, following the single purification step. Crystal screens were set up using the hanging drop method.

The top image shows the initial crystal hit and the bottom image shows optimized crystalization conditions. This demonstrates that biochemical homogeneity of the protein of interest can be a determining factor for crystallization success, and an optimized mammalian expression system produces proteins amenable for this crystallization. Further purification of nickel purified protein is readily done by size exclusion chromatography.

This is also a useful step towards evaluating protein production and optimizing conditions to yield properly folded proteins. The protein of interest should be the major species in the chromatographic elution, and the elution volume should correspond to the molecular weight of the protein.Earlier. EEU times may suggest aggregation or misfolding of the protein Once mastered.

This technique can be done in four days if performed properly. While performing this procedure, it's important to maintain sterile conditions for cell culture Following this procedure. Other methods like size, exclusion chromatography, multi-angle light scattering and differential scanning, fluorometry can be performed in order to assess the oli ization state and the thermal stability of the protein.