Roberts L M, Lamb F I, Pappin D J, Lord J M
J Biol Chem. 1985 Dec 15;260(29):15682-6.
A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.
用代表蓖麻毒蛋白B链中一种肽段所有可能序列的合成寡核苷酸混合物，筛选了一个用成熟蓖麻籽多聚腺苷酸（poly(A+)）RNA构建的cDNA文库。通过杂交选择出的八个最大的重组质粒，与一种单一的mRNA种类杂交，其翻译产物经免疫沉淀鉴定为前原凝集素。对这些克隆进行限制性内切酶分析表明，存在两类克隆，它们代表与两种不同但密切相关的前原凝集素mRNA种类互补的序列。其中一类克隆的cDNA核苷酸序列编码前原蓖麻毒素，该序列已在其他地方发表（Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265 - 270）。这里给出了第二类克隆的核苷酸序列，并表明其代表前原蓖麻凝集素。整个编码序列是从两个重叠的cDNA克隆推导出来的，这两个克隆的插入片段分别为1668和1151个碱基对。编码区定义了一种前原蛋白，其A链（266个氨基酸）之前有一个24个氨基酸的N端信号序列，A链通过一个12个氨基酸的连接肽与B链（262个氨基酸）相连。该蛋白被确认为蓖麻凝集素，因为推导的B链N端序列在蓖麻毒蛋白B链出现几个残基差异的区域，与纯化的蓖麻凝集素B链的序列完全一致。将蓖麻凝集素前体的核苷酸和推导的氨基酸序列与蓖麻毒素前体的序列进行了比较。
