cDNA cloning and functional expression of KM+, the mannose-binding lectin from Artocarpus integrifolia seeds
| Autor: | Maria Helena S. Goldman, Luis L. P. daSilva, Ademilson Panunto-Castelo, Jürgen Denecke, Jeanne B. Molfetta-Machado, Maria Cristina Roque-Barreira, Gustavo H. Goldman |
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| Rok vydání: | 2005 |
| Předmět: |
DNA
Complementary Molecular Sequence Data Biophysics Gene Expression Polymerase Chain Reaction Biochemistry law.invention Artocarpus law Amino Acid Sequence Cloning Molecular Molecular Biology Mannan-binding lectin Expression vector Base Sequence biology Mannose binding Lectin biology.organism_classification Molecular biology Recombinant Proteins Mannose-Binding Lectins Multigene Family Seeds biology.protein Jacalin Recombinant DNA Heterologous expression Plant Lectins |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1726:251-260 |
| ISSN: | 0304-4165 |
| DOI: | 10.1016/j.bbagen.2005.09.006 |
| Popis: | KM+, a mannose-binding lectin present in the seeds of Artocarpus integrifolia, has interesting biological properties and potential pharmaceutical use [A. Panunto-Castelo, M.A. Souza, M.C. Roque-Barreira, J.S. Silva, KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection, Glycobiology 11 (2001) 1035–1042. [1]; L.L.P. daSilva, A. Panunto-Castelo, M.H.S. Goldman, M.C. Roque-Barreira, R.S. Oliveira, M.D. Baruffi, J.B. Molfetta-Machado, Composition for preventing or treating appearance of epithelia wounds such as skin and corneal wounds or for immunomodulating, comprises lectin, Patent number WO20041008. [2]]. Here, we have isolated clones encoding the full-length KM+ primary sequence from a cDNA library, through matrix PCR-based screening methodology. Analysis of KM+ nucleotide and deduced amino acid sequences provided strong evidence that it neither enters the secretory pathway nor undergoes post-translational modifications, which is in sharp contrast with jacalin, the more abundant lectin from A. integrifolia seeds. Current investigations into the KM+ properties are often impaired by the difficulty in obtaining sufficient quantities of jacalin-free KM+ through direct seed extraction. To obtain active recombinant protein (rKM+) in larger amounts, we tested three different expression systems. Expression vectors were constructed to produce: (a) rKM+ in E. coli in its native form, (b) rKM+ with GST as an N-terminal tag and (c) native rKM+ in Saccharomyces cerevisiae. The presence of the GST-tag significantly improved the overall rKM+ yield; however, most of the obtained rGST-KM+ was insoluble. Production of rKM+ in the yeast host yielded the highest quantities of soluble lectin that retained the typical high-mannose oligosaccharide-binding properties of the natural protein. The possible biotechnological applications of recombinant KM+ are discussed. |
| Databáze: | OpenAIRE |
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