Abstract
BackgroundArtinM is a d-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system.ResultsThe ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized d-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure.ConclusionsOverall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.
Highlights
ArtinM is a D-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections
ArtinM is a D-mannose-binding lectin from seeds of Artocarpus integrifolia that stimulates macrophages and dendritic cells to produce IL-12 [6], an activity triggered by the ArtinM interaction with the Nglycans of TLR2 [7], and is able to induce Th1 biased immune response
The lectin ArtinM is capable of inducing neutrophil haptotactic migration mediated by the simultaneous interaction of its carbohydrate recognition domains (CRDs) with cell surface N-glycans [9] and extracellular matrix Nglycans [10,11,12]
Summary
ArtinM is a D-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. ArtinM is a D-mannose-binding lectin from seeds of Artocarpus integrifolia that stimulates macrophages and dendritic cells to produce IL-12 [6], an activity triggered by the ArtinM interaction with the Nglycans of TLR2 [7], and is able to induce Th1 biased immune response. The lectin ArtinM is capable of inducing neutrophil haptotactic migration mediated by the simultaneous interaction of its carbohydrate recognition domains (CRDs) with cell surface N-glycans (linked to the CXCR2 molecule) [9] and extracellular matrix Nglycans (linked to laminin) [10,11,12]. In this study we have aimed to produce highlevel expression of active soluble rArtinM in E. coli system
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