Scarafoni A, Carzaniga R, Harris N, Croy R R
Dipartimento di Scienze Molecolari Agroalimentari, Università degli Studi di Milano, Italy.
Plant Mol Biol. 2001 Aug;46(6):727-39. doi: 10.1023/a:1011675918805.
Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a 'hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.
napin是一种在油菜籽（甘蓝型油菜L.）及相关物种种子中发现的2S贮藏蛋白。利用蛋白质结构预测程序，我们在napin蛋白序列中鉴定出一个区域，该区域形成了一个“亲水环”，由位于蛋白质表面的氨基酸残基组成。针对该区域，我们构建了两个napin嵌合基因，其中包含肽激素亮氨酸脑啡肽的编码序列作为拓扑标记。一个版本具有包含连接子的11个氨基酸的单个脑啡肽序列，第二个包含该肽的串联重复序列，由22个氨基酸组成，插入到napin大亚基中。插入的肽序列改变了亲水氨基酸与疏水氨基酸的平衡，并为多肽链的该区域引入了灵活性。嵌合基因已在种子特异性napA基因启动子的控制下在烟草植株中表达。分析表明，工程化的napin蛋白在转基因种子的蛋白体中表达、运输、进行翻译后修饰并沉积，这表明改变后的napin蛋白与天然napin蛋白的行为方式相似。详细的免疫定位研究表明，肽序列的插入对napin蛋白在烟草种子蛋白体内的分布有显著影响。
