Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene.

Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene. Research Abstract Details 

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Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene. Abstract Text:

Tomomitsu Hatakeyama,Kouhei Shiba,Noriaki Matsuo,Tokiko Fujimoto,Tatsuya Oda,Hajime Sugawara,Haruhiko Aoyagi,

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.

Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene. Publishing Authors By Initials

T Hatakeyama,K Shiba,N Matsuo,T Fujimoto,T Oda,H Sugawara,H Aoyagi,

For similar investigative techniques: chemistry, analytical: crystallography: x-ray diffraction research abstracts see: investigative techniques: chemistry, analytical: crystallography: x-ray diffraction research

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Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 135

Page Numbers: 101-7

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Jan

YEAR: 2004

Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene. Information

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LANGUAGE: eng

NlmUniqueID: 376600

Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene. Keywords Mesh Terms:

KEYWORDS: X-Ray Diffraction

MESH TERMS: toxicity

Chemical & Substance for Abstract: Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene. Information

Substance Name: Acetylgalactosamine

Registry Number: 31022-50-1

Grant and Affiliation Information for Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene.

AFFILIATION: 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

Country: Japan

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MEDLINETA: J Biochem

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