Protein Purification
To establish cfIL-22 protein bioactivity, a 3-step purification process was developed that included ammonium sulfate precipitation, nickel affinity chromatography, and size exclusion chromatography. A representative western immunoblot tracks the protein through this purification scheme (Fig. 2.2A) to the final purified product (Fig. 2.2C). A corresponding Coomassie stained gel (Fig. 2.2B) shows the enrichment of this target protein applying this purification scheme. A representative Coomassie stain and silver stain of the final product (Fig. 2.2C) along with bioanalyzer assessment resulted in greater than a 90% purity for plant produced cfIL-22. While Table 2.1 shows cfIL-22 recovery is low (21%) indicating that optimization of the SEC step is needed, this purification scheme generated high quality protein at sufficient yield (5.4 mg/kg fresh tobacco leaf) for validating cfIL-22 bioactivity.