Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene.
CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+). Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I.
Department of Applied Chemistry, Faculty of Engineering, and Division of Biochemistry, Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521. thata@net.nagasaki-u.ac.jp
Tomomitsu
T
Hatakeyama
Kouhei
K
Shiba
Noriaki
N
Matsuo
Tokiko
T
Fujimoto
Tatsuya
T
Oda
Hajime
H
Sugawara
Haruhiko
H
Aoyagi
D000116:Acetylgalactosamine / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003460:Crystallization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005813:Genes, Synthetic; D006367:HeLa Cells; D006388:Hemagglutinins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D037181:Lectins, C-Type / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity; D014961:X-Ray Diffraction
D006388:Hemagglutinins; D037181:Lectins, C-Type; D011994:Recombinant Proteins; D000116:Acetylgalactosamine