title: Characterization and optimization of ArtinM lectin expression in Escherichia coli.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  Overall, 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.
authors.affiliation: Departamento de Biologia, FFCLRP, Av. Bandeirantes 3900, Ribeirão Preto 14040-901, Brazil. mcspranc@uft.edu.br
authors.forename: Maria-Cristina S
authors.initials: MC
authors.lastname: Pranchevicius
authors.forename: Leandro L
authors.initials: LL
authors.lastname: Oliveira
authors.forename: José C
authors.initials: JC
authors.lastname: Rosa
authors.forename: Nilton C
authors.initials: NC
authors.lastname: Avanci
authors.forename: Andréa C
authors.initials: AC
authors.lastname: Quiapim
authors.forename: Maria-Cristina
authors.initials: MC
authors.lastname: Roque-Barreira
authors.forename: Maria-Helena S
authors.initials: MH
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013696:Temperature
chemical_list: D037241:Mannose-Binding Lectins; D011994:Recombinant Proteins; D018664:Interleukin-12; D007544:Isopropyl Thiogalactoside