Characterization and optimization of ArtinM lectin expression in Escherichia coli

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.

ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation by Bacillus amyloliquefaciens SQR9

“…Expression and purification of the ResD and ResE proteins. The ResD and ResE proteins were expressed in E. coli BL21 carrying pET29a and constructs and were purified with Ni-nitrilotriacetic acid (NTA) agarose (Thermo Fisher Scientific, USA) (54,55). The plasmids (pET29a-resD and pET29a-resE) were constructed as follows.…”

The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties

“…IL-12 production also occurs when murine macrophages [72] are treated with native or recombinant forms of ArtinM. In addition, the recombinant form is able to induce the release of other inflammatory products, such as TNF-α and NO, at the same level as the native form (unpublished data).…”

Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

“…For mass spectrometry, the pull-down assay was repeated; however, the gel was staining with Comassie blue G-250, and the band related to the protein of S. venezuelensis that bound to the scFv was trypsinized and submitted to CID-MS/MS 30 (Quattro II, Micromass, Manchester, UK). Peptide fragments obtained were submitted and analyzed by BLAST ( http://www.ncbi.nlm.nih.gov/BLAST/ ) in relation to their similarity to proteins of Strongyloides sp.…”