Mouse anti-Human USP45 monoclonal antibody, clone 2I3

Oral delivery of insulin to diabetic patients is highly desirable because it would be non-invasive and more closely mimic normal physiology, but this route of administration typically results in low bioavailability due to low pH and enzymatic degradation along the gastrointestinal tract. To explore an alternative approach that may mitigate these obstacles and also facilitate local synthesis of new therapeutic protein molecules in the small intestine, we engineered the food-grade bacterium Lactococcus lactis (NZ9000) for nisin-inducible expression and secretion of a bioactive single-chain insulin (SCI) analog, SCI-57. We show that the addition of nisin during early-log phase has a modest inhibitory effect on cell growth but induction during mid-log phase has a negligible impact on proliferation, suggesting a tradeoff between cell growth rate and duration of induction. We find that a signal peptide such as usp45 is necessary for secretion of SCI-57 into the medium; furthermore, we demonstrate that this secreted SCI-57 is biologically active, as assessed by the ability of conditioned L. lactis medium to stimulate Akt signaling in differentiated 3T3-L1 adipocytes. Finally, we show that the biological activity of SCI-57 was enhanced by near-neutral or slightly alkaline pH during induction, which is comparable to the pH in the small intestine, and by removal of a C-terminal purification tag. This study demonstrates that food-grade bacteria can be engineered to secrete bioactive insulin analogs and opens up the possibility of oral insulin delivery using live microorganisms. Biotechnol. Bioeng. 2011;108: 1987-1996. (C) 2011 Wiley Periodicals, Inc.

Probiotics are beneficial microorganisms and have long been used in food production as well as health promotion products. Bioengineered probiotics are used to express and transfer native or recombinant molecules to the mucosal surface of the digestive tract to improve feed efficiency and promote health. Lactococcus lactis is a potential probiotic candidate to produce useful biological proteins. The aim of this investigation was to develop a recombinant Lactococcus lactis with the potential of producing phytase. To enhance the efficiency of expression and secretion of recombinant phytase, usp45 signal peptide was added to the expression vector containing phytase gene (appA2) derived from Escherichia coli. Sequencing of recombinant plasmid containing appA2 showed the correct construction of plasmid. Total length of the phytase insert was 1.25kbp. A Blast search of the cloned fragment showed 99% similarity to the reported E. coli phytase sequence in the GenBank (accession number: AM946981.2). A plasmid containing usp45 and appA2 electrotransferred into Lactococcus lactis. Zymogram with polyacrylamide gel revealed that the protein extract from the supernatant and the cell pellet of recombinant bacteria had phytase activity. Enzyme activity of 4U/ml was obtained in cell extracts, and supernatant maximal phytase activity was 19U/ml. The recombinant L. lactis was supplemented in broiler chicken feed and showed the increase of apparent digestibility on phytate phosphorus in the digestive tract and it was same as performance of E. coli commercial phytase.