Recombinant Lactococcus Lactis subsp. lactis Bacteria for Oral Delivery of GLP-1
Payal Agarwal, Doctoral Fellow, St. John’s University
Blasé Billack, Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences
Jun Shao, Department of Pharmacy and Administrative Sciences, College of Pharmacy and Health Sciences
Abstract: Purpose: The objective of the present study was to construct a genetically modified Lactococcus lactis (L. lactis) bacterium transformed with recombinant plasmid for the oral delivery of Glucagon like peptide-1 (GLP-1) for the treatment of type-2 diabetes mellitus.
Methods: Construction of recombinant plasmid was carried out using a pUB1000 as an expression vector. GLP-1 c-DNA was synthesized as a fusion gene consisting of mature part of the peptide fused to the two restriction sites SalI and BamHI. Polymerase Chain Reaction was used to amplify the GLP-1 c-DNA. Purified c-DNA was ligated into the expression cassette of pUB1000 downstream to usp45 signal peptide to get recombinant plasmid pUBGLP-1. The ligated construct (pUBGLP-1) was verified using restriction enzyme digestion and gene sequencing. Finally, pUBGLP-1 was transformed into the competent L. lactis cells by electroporation. The transformed bacterial cell colonies were selected and characterized for the production and secretion of GLP-1 into the culture media. Bioactivity of the secreted GLP-1 was confirmed by insulinotropic activity on pancreatic cell line (HIT-T15). The in vitro transport of GLP-1 by the transformed L. lactis through MDCK cell monolayer was studied to evaluate the potential in vivo absorption efficacy.
Results: pUBGLP-1 plasmid was successfully constructed. It was observed that the recombinant L. lactis containing pUBGLP-1 could efficiently secrete biologically active GLP-1 (7-36) amide in the culture media, about 60 pM in 12 hr. It was shown that there was a significant increase (~ 2 fold) in the insulin release by HIT-T15 cells in the presence of the recombinant L. lactis as compared to the untransformed L. lactis. The transport studies show that there was a significant increase (~ 2.5 fold) in the transport rate of GLP-1 by L. lactis delivery as compared to the free solution form.
Conclusion: A GLP-1 secreting L. lactis was constructed, which could increase the absorption of GLP-1 in vitro. Future in vivo study will further prove the applicability of this system.