Measurement
Protein Yield determination is necessary for quantifying the efficiency of our purification proposed system and in our case require the quantification of both starch, to which our protein is bounded, and our target protein (GFP).
Therefore, we collected the leaves at the end of the light period, blended them to brake the cells and liberate the starch granules, which contain our protein attached to GBSS. After that, we filtered and centrifuged the extract to separate the starch granules from the rest of the cell components and obtain the purified starch.
Starch purification from leaves of Arabidopsis thaliana expressing GBSS-Xa-GFP (BBa_K3045002). Leaves were homogenized with a waring blender, the homogenate was filtered through two layers of Miracloth and after centrifugation the supernatant was removed and the pellet contained intact starch granules.
One fraction of the purified starch will be used for starch quantification. The rest of the purified starch will be used for protein purification and quantification.
Starch quantification
As starch is a complex structure of glucose molecules (as shown in Figure Structure of the starch granule) starch quantification was determined as the amount of glucose units present in the purified starch fraction. So the first step was to brake the starch granules into glucose-chains by alkaline hydrolysis. Then, using an enzymatic starch assay kit (R-biopharma), glucose units were liberated and quantified using two coupled-reactions in which NAD is reduced to NADH, a molecule that can be quantify by measuring the absorbance at 340 nm (as shown in Figure Starch quantification protocol). The resulting absorbance were transformed in glucose molecule amount using a calibration curve (as shown in Figure Glucose calibration curve). The plot represents the absorbance at 340 nm versus glucose concentration (µM). With the formula obtained we calculated how much starch (as glucose molecules) we were able to purify from our plants.
Structure of the starch granule. Starch granules are composed of a-1,4-glucans with some a-1,6-branches, placing them parallel to each other, allowing double helix formation. The branches are specifically placed, giving regions of branching, known as amorphous lamella, and regions of exclusively linear chains which form crystalline lamella, and these lamella form defined growth rings. Specific enzymes are needed to synthesize and degrade this highly ordered insoluble structure. (O´Neill and Field, 2015). After that, glucose concentration was transformed in mg of starch using glucose molecular weight.
Starch quantification protocol based in the enzymatic assay kit from R-Biopharma.
Glucose calibration curve used for starch quantification.
Protein purification and quantification
To know the amount of pure GFP protein obtained in our experiment we used the technique of Western blotting. This technique consists in separating the proteins by electrophoresis in a polyacrylamide gel and then transfer the proteins to a membrane. Proteins are detected with specific antibodies and then a labeled secondary antibody is added, which binds to the primary antibody and it is colorimetric detected.
The quantity of GFP protein detected in the western blot was quantified by densitometry using the software ImageJ. Standard amounts of GFP and different amounts of purified GFP were load in the western blot. A plot with a linear regression was generated using the standars GFP.
The plot represent the signal intensity versus ng of GFP. With the formula obtained we could calculate how much GFP we were able to purify from our plants.
Protein Yield
Protein yield is the efficiency marker of the proposed model for protein production. It indicates the amount of protein produce per kilogram of leaves. Therefore, using the amount of starch determined as described in “starch quantification” and the amount of GFP recovered as described in “protein purification and quantification” we will determine the Protein Yield as follow:
Protein Yield (µg/Kg leaves) = (g starch/Kg leaves) x (µg protein/g starch)