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.