Protein Characterization
Qualitative and quantitative assessment of the purified protein included SDS-PAGE (NuPage 12% Bis-Tris; Invitrogen) resolution and detection by either anti-His (Genscript) western immunoblotting, silver staining (Pierce) or Coomassie Simply Blue Safe Staining (Invitrogen). Purified protein was also subject to a bioanalyzer (Experion, Pro260 chip, Bio-Rad) in providing an additional measure of purity and concentration. Spectrophotometry using a Nanodrop 1000 and the protein extinction coefficient (14,940 M-1cm-1) was also used to confirm protein concentration. Endotoxin levels of SEC-purified cfIL-22 and negative control (pBK equivalent) were determined by the Limulus Amoebocyte Assay chromogenic method in accordance with manufacturer’s instructions (Charles River, Endosafe Kinetic Chromogenic Limulus Amebocyte Lysate Endochrome-K kit). Purified protein was further analyzed by MS-MS analysis at the University of Arkansas Medical Sciences (UAMS) Proteomics Core Facility and N-terminal sequencing by the Protein Facility (Office of Biotechnology, Iowa State University). N-glycan digest was performed using PNGase F (New England Biolabs) according to manufacturer’s guidelines. Amino acid sequence was submitted both to I-TASSER and Raptor X modeling programs for structure prediction.