title: Stability of recombinant human alpha-1-antitrypsin produced in rice in infant formula.
abstract: Human milk contains several biologically active proteins that benefit the breast-fed infant. In order to survive in the gastrointestinal tract, these proteins need to be protected against proteolysis. Since human milk contains relatively high concentrations of alpha-1-antitrypsin (AAT), we have expressed recombinant AAT in rice to explore the possibility of supplementing infant formula with this protein. The stability of recombinant AAT was examined by biochemical and functional assays, such as SDS-PAGE, Western blotting, ELISA, elastase and trypsin inhibition, following exposure to heat, low pH, and in vitro digestion, conducted in both phosphate buffered saline and infant formula. Native AAT is resistant to acidic environments down to pH 2 for 1 h and can withstand in vitro digestion modeled after conditions in the infant gut. Recombinant AAT is nearly as resistant as the native form in buffer, and is equally resilient in formula. Heat treatments (60 degrees C for 15 min, 72 degrees C for 20 sec, 85 degrees C for 3 min, and 137 degrees C for 20 sec) revealed that recombinant AAT is not as stable as native AAT in buffer, particularly at higher temperatures. While significantly less recombinant AAT is detected by ELISA after heating in formula, addition of bile extract can restore epitopes resulting in higher concentrations, suggesting protein aggregation that may not affect AAT activity. This study shows that recombinant AAT may survive the conditions of the infant stomach and duodenum and affect protein digestion in the infant small intestine.
authors.affiliation: Department of Nutrition, University of California, Davis 95616, USA.
authors.forename: Winyoo
authors.initials: W
authors.lastname: Chowanadisai
authors.forename: Jianmin
authors.initials: J
authors.lastname: Huang
authors.forename: Ning
authors.initials: N
authors.lastname: Huang
authors.forename: Bo
authors.initials: B
authors.lastname: Lönnerdal
mesh_terms: D015153:Blotting, Western; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007223:Infant; D007225:Infant Food / Q000032:analysis*; D012275:Oryza / Q000235:genetics*; D010194:Pancreatin / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D010194:Pancreatin; D010434:Pepsin A