We investigated how to extract recombinant proteins from plant cells. More officially, we answered the question of whether the apoplast protein extraction method is feasible when applied to the recombinant proteins. This extraction method checks proteins secreted into the apoplast, so they have higher homogeneity.
There is no need for cell destruction, which reduce the interference from intracellular proteins and the cell subsequent purification costs. We will focus on how to increase the secretion of recombinant proteins in the upper plant space to further improve the efficiency of the apoplast protein extraction method. To observe subcellular localization of GFP after 48 hours of agroinfiltration of Nicotiana benthamiana, select a flat leaf and make a six-millimeter hole using a hole punch.
Drip 30%sucrose solution onto a glass slide. Place the leaf sample with the back facing upwards and cover it with a cover glass. Collect the intact, fresh N.benthamiana leaves expressing recombinant GFP at three days post agroinfiltration and weigh the leaves.
Rinse the leaf blade with pre-cooled sterilized double-distilled water to remove debris. Submerge 20 grams with the abaxial side down in 500 milliliters of pre-cooled 100 millimolar phosphate buffer in a 1, 000 milliliter wide mouth beaker. Cover the leaves with 100 mesh nylon fine mesh yarn and a plastic plate.
Connect the beaker to a vacuum pump. Apply 0.8 megapascal vacuum for one minute, then quickly restore to atmospheric pressure. Remove leaves from the buffer and gently blot dry with absorbent paper.
Roll up the leaves and wrap them with mesh yarn. For collecting apoplastic washing fluid, or AWF, position rolled leaves tip side up in 50 milliliter centrifuge tubes with mesh yarn fastened by the tube caps. Centrifuge the leaves at 500 G for five minutes at four degrees Celsius.
Remove the leaves and collect AWF using a pipette. Add nickel Sepharose excel resin to a tube in the one to 1, 000 ratio relative to the protein extract volume. Wash nickel resin three times with 10 times the resin volume of double-distilled water and phosphate buffer, separately in turn.
Incubate protein extract with nickel resin for two hours to allow full binding, then, centrifuge at 500 G for five minutes at four degrees Celsius to remove unbound proteins in the supernatant. Wash three times with 20 times the resin volume of phosphate buffer. After the last wash, add 10 times the resin volume of 250 millimolar imidazole to elute GFP His.
Centrifuge the mixture at 500 G for five minutes at four degrees Celsius and collect the supernatant. Western blot analysis confirmed the transient expression of 30 kilodalton recombinant protein GFP His in N.benthamiana. Fluorescence microscopy illustrated the presence of GFP His within the apoplast.
AWF proteins extracted by vacuum infiltration centrifugation also contain GFP His. Out of the eight extraction solutions tested, phosphate buffer extracted the greatest amount of AWF and GFP His. Comparing phosphate buffer at varying pH revealed improved AWF and GFP His extraction at neutral to mildly alkaline conditions.
Additionally, 100 millimolar phosphate buffer showed maximal efficacy for recovering AWF and GFP His. With a 100 millimolar phosphate buffer, 495 micrograms of total AWF proteins per gram of fresh leaf were recovered from the apoplast. The extracted AWF contained 10 micrograms of GFP His, which corresponded to approximately 18%of the GFP His in the total soluble protein.
Following nickel affinity chromatography, GFP His purity reached 84.3%from AWF extraction, substantially higher than the 44.9%purity achieved through total soluble protein extraction.
