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

Biochimica Et Biophysica Acta
Tomomitsu HatakeyamaKohei Shiba

Abstract

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...Continue Reading

References

Jul 1, 1995·Bioscience, Biotechnology, and Biochemistry·T HatakeyamaN Yamasaki
Oct 6, 1999·Current Opinion in Structural Biology·K Drickamer
Sep 12, 2002·Biochimica Et Biophysica Acta·David C Kilpatrick
Jul 28, 2004·Journal of Molecular Biology·Katsunori HoriiHiroshi Mizuno
Feb 17, 2005·Journal of Biochemistry·Takuya KuramotoTatsuya Oda

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Citations

Jan 21, 2011·The Journal of Biological Chemistry·Tomomitsu HatakeyamaHideaki Unno
Dec 9, 2008·Fish & Shellfish Immunology·Ying ZhangBingxin Huang
Oct 11, 2012·Bioscience, Biotechnology, and Biochemistry·Tomomitsu HatakeyamaShuichiro Goda

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