Characterization of a recombinant C-type lectin, rCEL-IV, expressed in Escherichia coli cells using a synthetic gene.
The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca2+-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 alpha-galactosides than for beta-galactosides, while other C. echinata lectins show preferential binding of beta-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.
Hatakeyama
Tomomitsu
T
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. thata@net.nagasaki-u.ac.jp
Hozawa
Takao
T
Hirotani
Iyo
I
Tsuda
Nobuaki
N
Kusunoki
Masami
M
Shiba
Kohei
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005813:Genes, Synthetic / Q000235:genetics; D006386:Hemagglutination Tests; D037181:Lectins, C-Type / Q000235:genetics*; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D012616:Sea Cucumbers / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C095642:CEL-IV, Cucumaria echinata; D037181:Lectins, C-Type; D011994:Recombinant Proteins