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
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DOI：
10.1093/jb/mvh012
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发表时间：
2004-01-01
影响因子：
2.7
通讯作者：
Aoyagi, H
中科院分区：
文献类型：
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作者：
Hatakeyama, T;Shiba, K;Aoyagi, H
celi是一种c型凝集素，从细瓜科植物黄瓜中分离得到。该凝集素具有很高的n -乙酰半乳糖胺结合特异性。利用人工合成的寡核苷酸组合构建了一个编码重组cel - 1的人工基因（rcel - 1），并在大肠杆菌细胞中表达。由于获得的重组蛋白是包涵体，包涵体用尿素和2-巯基乙醇溶解，蛋白在纯化和透析过程中被重新折叠。纯化的cel - 1在相对较高的Ca2+浓度下显示出与天然cel - 1相当的血凝活性，而在较低的Ca2+浓度下，由于Ca2+结合亲和力降低，其血凝活性较弱。通过血凝抑制试验，rcel - 1表现出与天然cel - 1相似的碳水化合物结合特异性，包括较强的galnac结合特异性。重组蛋白与天然蛋白的远紫外- cd光谱比较表明，这两种蛋白在与Ca2+结合后发生了相似的构象变化。在与天然蛋白相同的条件下也获得了rcel - 1的单晶，表明它们具有相似的三级结构。虽然天然cel - 1对培养细胞表现出很强的细胞毒性，但rcel - 1表现出较低的细胞毒性。这些结果表明，rcel - 1具有与天然cel - 1相似的三级结构和碳水化合物结合特异性。然而，这两种蛋白质之间的性质有细微的差异，这可能是由于rcel - 1的n端有额外的蛋氨酸残基。
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 Ca2+-concentrations, whereas it was weaker at lower Ca2+-concentrations due to decreased Ca2+-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 Ca2+. 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.