Cloning and Production of cfIL-22
The cfIL-22 gene sequence (generously provided by Dr. Sylvie Quiniou) was synthesized as the native sequence as well as codon optimized for enhanced production based on codon preference in the plant host system,Nicotiana benthamiana . Plant expression constructs were created by transferring the gene cassettes into a pBIBKan (pBK) expression vector under the control of a constitutive promoter (35S duel enhanced CaMV), tobacco etch virus (TEV) translational enhancer and Tnos terminator (Figure 2.). Both constructs used sequences encoding the native catfish IL-22 signal peptide for trafficking through the endomembrane system.
As determining expression kinetics in planta is protein specific and important for maximizing protein yield, leaf tissue from infiltrated plants collected at 48, 72, 96, and 120 hours post-infiltration was analyzed. Relative comparison of protein quality and yield were compared by anti-His western blot analysis with maximum cfIL-22 protein accumulation at 96 hours post infiltration (Supporting material Figure S2). The cfIL-22 product corresponds to a 20 and 25kDa product on western blot. As plant transient expression of both native and codon-optimized cfIL-22 constructs were qualitatively and quantitatively comparable (Supporting material Figure S3), all further studies used the construct expressing the native coding sequence (cfIL-22). As determining optimal extraction buffer is also protein dependent, several buffers were tested; acidic (sodium citrate buffer, pH 5), neutral (plant phosphate buffer, pH 7), and basic (Tris buffer, pH 8.5) ranges. Plant expressed cfIL-22 protein preferentially extracted in the higher pH buffer (Supporting material Figure S4) and was adopted for all analyses in this study.