Hagelin K, Rodriguez-Suarez R, Katzen F, Wolosiuk R A, Baldi P C, Giambartolomei G H, Fossati C A, Dyer T
Instituto de Investigaciones Bioquimicas Luis F. Leloir Fundación Campomar, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Cell Mol Biol (Noisy-le-grand). 1996 Jul;42(5):673-82.
The primary structure of several chloroplast fructose-1,6-bisphosphatase (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally. We analyzed whether CFBPases from different phylogenetic origin contain a common epitope. To this end a DNA fragment of 1200 base pairs encoding 338 amino acid residues of wheat CFBPase (38 kDa) was cloned in the expression plasmid pGEX-1 in frame with the gene coding for glutathione S-transferase (GT) of Schistosoma japonicun (26.5 kDa). Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyranoside, centrifugation of the lysate partitioned 10% of the fusion protein in the supernatant fraction and the remaining 90% in the precipitate. The expected 65 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glutathione-agarose. This fusion protein was successfully used to produce a monoclonal antibody that specifically recognized the CFBPase region of the fusion protein but not the GT moiety. Moreover, the monoclonal antibody immunoreacted not only with polypeptides (ca. 40 kDa) present in leaf crude extracts of other varieties of wheat (Triticum spelta, T. aestivum and T. durum), but also with homogeneous preparations of the spinach (Spinacia oleracea) and rapeseed (Brassica napus) enzymes. Thus, the cross reaction of this monoclonal antibody with counterparts from different plant species indicates the persistency of a common epitope through biological evolution.
几种叶绿体果糖-1,6-二磷酸酶（CFBPase）的一级结构已根据DNA序列推导得出，但只有菠菜、豌豆和油菜的该酶在结构上得到了表征。我们分析了来自不同系统发育起源的CFBPase是否含有共同的表位。为此，将编码小麦CFBPase（38 kDa）338个氨基酸残基的1200个碱基对的DNA片段克隆到表达质粒pGEX-1中，与日本血吸虫谷胱甘肽S-转移酶（GT，26.5 kDa）的编码基因读框一致。用异丙基-β-D-硫代半乳糖苷转化大肠杆菌并诱导后，裂解物离心，10%的融合蛋白在上清液部分，其余90%在沉淀中。通过谷胱甘肽琼脂糖柱亲和层析从可溶性部分和颗粒部分纯化出预期的65 kDa蛋白。该融合蛋白成功用于制备单克隆抗体，该抗体特异性识别融合蛋白的CFBPase区域，但不识别GT部分。此外，该单克隆抗体不仅与其他小麦品种（斯卑尔脱小麦、普通小麦和硬粒小麦）叶片粗提物中的多肽（约40 kDa）发生免疫反应，还与菠菜（菠菜）和油菜（甘蓝型油菜）酶的纯品发生免疫反应。因此，这种单克隆抗体与不同植物物种对应物的交叉反应表明，共同表位在生物进化过程中持续存在。
