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.