Trypsin spin column

Wt 5×GLP-1 and trp 5×GLP-1 positive controls were expressed in E. coli with a C-terminal His-tag and purified to approximately 85% purity (GenScript, Piscataway, NJ). The Lactobacillus-produced Lp wt 5×GLP-1 and Lp trp 5×GLP-1 were purified from the culture supernatant of strains (KKA405 and KKA394) with anti-E-tag monoclonal antibodies coupled to an NHS-HiTrap sepharose column (GE-healthcare) according to the manufacturer’s instructions. The eluate was concentrated to a volume of 0.5 ml using an Amicon Ultra-4 3K centrifugal filter (Millipore, Billerica, USA). The concentration of purified 5×GLP-1 was determined by the Micro BCA Protein Assay Kit (Pierce, Rockford, USA) with E. coli produced 5×GLP-1 as a standard.
Both E. coli- and Lactobacillus-produced 5×GLP-1 were subjected to in vitro trypsin digestion to mirror the in vivo situation. 5×GLP-1 was digested for 7 min at 37°C using a Trypsin Spin Column (TT0010, Sigma) according to the manufacturer’s instructions. Tryptic digested proteins were analyzed by silver staining before use in the in vitro experiments.

To confirm the identity of the expressed and purified protein, mass spectrometry was performed using a method described previously [20 (link)]. The proteins were digested using a trypsin spin column (Sigma, St. Louis, MO, USA), prepared according to the manufacturer’s instructions. Using an LTQ Orbitrap Elite (Thermo Scientific, Waltham, MA, USA) with a nano ESI interface and Ultimate 3000 RSLC nano-HPLC, eluted peptides were analysed at Bio21 Institute (Parkville, Australia). The obtained spectra were identified using Mascot search engine (Matrix Science, Boston, MA, USA) with our in-house database of the oyster proteome (https://www.uniprot.org/proteomes/UP000005408 (accessed on 12 April 2021), and in addition the common Repository of Adventitious Proteins sequences (https://www.thegpm.org/crap/ (accessed on 15 April 2019).