A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

The overall goal of this procedure is to produce high yields of folic glycosylated and enzymatically active granzymes in human embryonic kidney 2 93 T cells. This is accomplished by first expanding HEC 2 93 T cells in appropriate tissue culture dishes. The usual preparation size after the expansion consists of 20 to 25 culture dishes.

The second step is to transiently transfect the expanded cells with a granzyme expression plasmid using the efficient calcium phosphate method. Next, the cell supernat are harvested and the secreted grand enzymes are purified by immobilized metal affinity chromatography. The final steps are to activate the grand enzymes by enter kinase treatment and to further purify the proteases via cation exchange chromatography.

Ultimately, enzymatic activity is tested using a rapid and reliable chole metric assay As the availability of active protease in sufficient amounts is a limiting factor in granzyme research. This method can help answer key questions in the field, such as characterization of new cell death pathways in different organisms, or identification of novel protease substrates in whole proteome approaches To express grand enzymes. First grow HEC 2 93 T cells in 20 milliliters of culture medium, using 150 by 25 millimeter tissue culture dishes split the cells at 80%Co fluency cells loosely attach, but can be mechanically detached without trypsin by rigorously pipetting up and down plate.

The cells the night before transfection to give 60 to 70%co fluency at the day of transfection. A usual preparation size consists of 20 to 25 culture dishes. One hour prior to transfection changed the standard culture medium to 20 milliliters of transfection medium near the end of the one hour incubation step mixed 400 micrograms of plasma DNA with 10.95 milliliters of double distilled water and 1.55 milliliters of two molar calcium chloride in 50 milliliter tubes one to two minutes prior to the transfection.

At 12.5 milliliters of two x heaps buffered saline to 12.5 milliliters of the DNA calcium solution mixed by inversion of the tubes before incubating for 30 seconds. At room temperature, add this mixture, dropwise directly to the cells at five milliliters per dish, sprinkle evenly over the entire area, turning the medium to a slightly orange color. Incubate the culture dishes for seven to 11 hours in a tissue culture incubator.

After the incubation, a fine precipitate is visible. Remove the transfection medium. Rinse carefully with prewarm PBS and add 20 milliliters of serum free culture medium before incubating for 72 hours in a tissue culture incubator, analyze the transfection and expression efficiency by running a sample of the cell supernatant on SDS page and staining with kumasi.

Brilliant blue. To visualize a granzyme band. After the incubation decant the cell culture supernatants into 250 milliliter tubes and clear by centrifugation.

Carry out a first spin that will clear the medium from the detached cells. Transfer the supernatant into fresh 250 milliliter tubes and spin at 4, 000 Gs for 30 minutes at four degrees Celsius to remove any remaining cell debris. Next, add five milliliters of sodium chloride, 6.25 milliliters of tris base and one milliliter of nickel sulfate plus 250 milliliters of cleared supernatant.

Filter the supernatant using a 500 milliliter vacuum filter unit. Equilibrate a nickel immobilized metal ion affinity chromatography column with his binding buffer. A using a suitable FPLC system.

Then apply the cleared supernatant to the equilibrated column at a flow rate of 0.5 milliliters per minute at four degrees Celsius. After the supernatant is applied, wash the column with his binding buffer until the ultraviolet or UV absorbent baseline is reached. Elute the granzymes with a linear 20 milliliter ILE gradient at a flow rate of 0.5 milliliters per minute while collecting two milliliter fractions.

Analyze the elucian fractions by SDS page and kumasi staining. Pull the grand zyme containing fractions in a dazing tube. Store a small sample at minus 20 degrees Celsius as the pre entero kinase control.

Add entero kinase at 0.02 units per 50 milliliters of initial supernatant directly to the pooled fraction in the dialysis tube and dialyze overnight at room temperature in entero kinase buffer. The next day analyze kinase treated grand in comparison to the pre enterer kinase control by SDS page and kumasi blue staining. When end terminal processing is complete.

Change the dialysis buffer to S buffer A and dialyze for another four hours at room temperature before filtering the DIALATE eight. Next, equilibrate an S column with S buffer. A.Load the sample on the column at a flow rate of 0.5 milliliters per minute at four degrees celsius after sample loading.

Wash the column with S buffer A until UV absorbent baseline is reached. Elute the granzymes with a 30 milliliter linear sodium chloride gradient granzyme a elutes at approximately 650 millimolar sodium chloride granzyme B at approximately 700 millimolar sodium chloride and granzyme M at approximately 750 millimolar sodium chloride. Pool the fractions containing granzymes and concentrate to about 100 micromolar in spin filters, aliquot the concentrated granzyme preparations and store at minus 80 degrees Celsius.

Next, analyze elucian fractions by SDS page and chole metric assays to perform the chole metric assays distribute small granzyme samples from ellucian fractions or concentrated stocks in 96. Well flat bottom plates include a positive control and a negative control. Add 100 microliters of assay buffer to each.

Well incubate the plate for 10 minutes at 37 degrees Celsius. Finally, measure the optical density at 405 nanometers in a microplate reader. This protocol will produce high yields of pure granzymes as demonstrated on this representative kumasi stained SDS page gel showing granzyme BE from the S column.

The recombinant granzymes display similar activity as native protease preparations as shown in this color metric assay comparing recombinant and native granzyme B activity. Importantly, endo H treatment indicates that the granzymes produced in HEC 2 93 T cells are fully glycosylated. The recombinant grand enzymes are capable of cleaving macromolecular protein substrates as demonstrated in this representative granzyme a cleavage assay using the bacterial protein substrate and UOCD.

Here, the band representing cleaved N-O-U-C-D slowly disappears with increasing granzyme concentration. After watching this video, you should have a good understanding of how to produce fully glycosylated and pro active grand enzymes at high yields in HK 2 93 T cells.