Dataset Information

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus.

ABSTRACT: Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFN?) was expressed in insect larvae that constitute important agronomic plagues. rFeIFN? accumulated in the hemolymph of Spodoptera frugiperda larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFN? was recovered after purification with a specific activity of 1?×?106 IU mg-1. By this method, we obtained 8.9?×?104 IU of purified rFeIFN? per larva. The product was biologically active in vitro, with an antiviral activity of 9.5?×?104 IU mL-1, as well as a potent antitumor activity comparable to that of the commercial FeIFN?. The glycosylation of rFeIFN? was confirmed by peptide-N-glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFN? production in laboratory research or veterinary medicine applications.

SUBMITTER: Arregui MB

PROVIDER: S-EPMC6063813 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus.

Arregui Mariana Bernadett MB Mc Callum Gregorio Juan GJ Smith Ignacio I Villaverde Marcela Solange MS Wolman Federico Javier FJ Targovnik Alexandra Marisa AM Miranda María Victoria MV

3 Biotech 20180727 8

Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNβ) was expressed in insect larvae that constitute important agronomic plagues. rFeIFNβ accumulated in the hemolymph of <i>Spodoptera frugiperda</i> larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNβ w ...[more]

PMID: 30073126

Similar Datasets

Project description:BackgroundLeukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli).ResultsThe BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity.ConclusionsThe present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.

Dataset Information

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus.

Publications

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets