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 Allied Health
Professions
Jun Shao, Department of Pharmacy and
Administrative Sciences, College of Pharmacy and Allied Health
Professions
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.