Characterization of a recombinant C-type lectin, rCEL-IV, expressed in Escherichia coli cells using a synthetic gene

• Published in: Biochimica et biophysica acta Vol. 1760; no. 3; pp. 318 - 325
• Main Authors: Hatakeyama, Tomomitsu, Hozawa, Takao, Hirotani, Iyo, Tsuda, Nobuaki, Kusunoki, Masami, Shiba, Kohei
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2006
• Subjects: Amino Acid Sequence; Animals; Artificial gene; Base Sequence; C-type lectin; Circular Dichroism; Crystal; Crystallization; Cucumaria echinata; Dynamic light scattering; Escherichia coli - genetics; Escherichia coli - metabolism; Expression; Genes, Synthetic - genetics; Hemagglutination Tests; Lectins, C-Type - genetics; Marine invertebrate; Molecular Sequence Data; Protein Structure, Quaternary; Rabbits; Recombinant Proteins - chemistry; Sea Cucumbers - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; CD; Marine invertebrate; TBS; MALDI-TOF; rCEL-IV; DLS; EDTA; nCEL-IV; RE; CRD; Dynamic light scattering; Expression; CTLD; Artificial gene; C-type lectin; Cucumaria echinata; Crystal
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2006.01.011

The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca 2+-dependent galactose-specific lectins. One of these lectins, CEL-IV, is composed of a C-type carbohydrate-recognition domain homotetramer. CEL-IV exhibits higher specificity for α-galactosides than for β-galactosides, while other C. echinata lectins show preferential binding of β-galactosides. We constructed an artificial synthetic gene for recombinant CEL-IV (rCEL-IV) based on the amino acid sequence previously determined from the purified protein. rCEL-IV was expressed in Escherichia coli cells as inclusion bodies. After the refolding process, most of rCEL-IV spontaneously formed a homotetramer structure having interchain disulfide bonds. The secondary structure of rCEL-IV was similar to that of the native one, as judged by the comparison of the far UV-circular dichroism spectra of rCEL-IV and native CEL-IV (nCEL-IV). Carbohydrate-binding specificity of rCEL-IV was confirmed to be similar to that of nCEL-IV from the results of the binding-inhibition assay using liposomes composed of rabbit erythrocyte lipids. Crystals of rCEL-IV were obtained in a few days by the sitting drop vapor diffusion method. These results indicate that rCEL-IV achieved essentially correct three-dimensional structure, including the carbohydrate-binding sites, and it would be very useful for further study on the carbohydrate-recognition mechanism by mutational and X-ray crystallographic analyses.