Jia S, Mee R P, Morford G, Agrwal N, Voss P G, Moutsatsos I K, Wang J L
Department of Biochemistry, Michigan State University, East Lansing 48824.
Gene. 1987;60(2-3):197-204. doi: 10.1016/0378-1119(87)90228-9.
Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a lambda gt 11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing beta-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by affinity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against beta-galactosidase. These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli. Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.
针对碳水化合物结合蛋白35（CBP35，一种半乳糖特异性凝集素）的亲和纯化抗体，被用于筛选源自3T3成纤维细胞mRNA的λgt 11表达文库。该筛选产生了几个含有CBP35 cDNA的推定克隆，其中一个根据其表达含β-半乳糖苷酶和CBP35序列的融合蛋白进行了表征。对含有融合蛋白的裂解物进行有限蛋白酶解，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳并用抗CBP35进行免疫印迹，得到的肽图谱与对真实CBP35进行平行处理所获得的图谱相当。这种有限蛋白酶解后，在与半乳糖偶联的琼脂糖柱上进行亲和层析，也产生了一种具有碳水化合物结合活性的30 kDa多肽。该多肽可用抗CBP35进行免疫印迹，但不能用针对β-半乳糖苷酶的抗体进行免疫印迹。这些结果表明，我们已经鉴定出一个CBP35的cDNA克隆，其产生在大肠杆菌中产生的具有凝集素活性的重组多肽。使用该cDNA克隆作为探针，Northern印迹分析表明，当静止的3T3细胞通过添加血清生长因子被激活时，CBP35基因的表达增加。
