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.