Xu Hui, Montoya Fernando U, Wang Zeping, Lee James M, Reeves Raymond, Linthicum D Scott, Magnuson Nancy S
Department of Chemical Engineering, Washington State University, Pullman, WA 99164, USA.
Protein Expr Purif. 2002 Apr;24(3):384-94. doi: 10.1006/prep.2001.1580.
In order to facilitate production and secretion of a soluble form of a small, single-chain antibody ScFv (32 kDa) in tobacco cell suspension culture, several modifications were made simultaneously to the antibody cDNA that included elements that have been shown to regulate the expression of proteins in plants. The scFv cDNA was initially ligated into a binary vector under the control of the CaMV 35S promoter and the T7 terminator for expression in tobacco suspension culture. Subsequently, modifications were engineered into the cDNA for enhancement of scFv production. These included the following: (i) the signal peptide (SP) of the tobacco pathogenesis-related protein PR1a which was added in-frame to the N-terminal end of scFv cDNA; (ii) a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence), which was fused to the N-terminal end of the SP; and (iii) the endoplasmic reticulum retention signal peptide KDEL, which was added to the C-terminal end of the scFv protein. Using a modified disruption method involving pectinase, the highest expression of total scFv (344 ng scFv/g cell) occurred when the plant leader sequence, the TEV sequence, and the KDEL peptide were all present in the expression construct. Although the addition of the KDEL sequence significantly increased the total yield of protein 5.4-fold, it did not increase the overall amount of protein secreted. These studies indicate that while the SP is very important in promoting secretion of the scFv, it had little influence on increasing scFv secretion levels even when both the TEV and the KDEL sequences significantly increased overall protein levels.
为了便于在烟草细胞悬浮培养物中生产和分泌可溶性形式的小单链抗体ScFv（32 kDa），对抗体cDNA进行了多项同时修饰，其中包括已被证明可调节植物中蛋白质表达的元件。scFv cDNA最初被连接到一个二元载体中，在花椰菜花叶病毒（CaMV）35S启动子和T7终止子的控制下，用于在烟草悬浮培养物中表达。随后，对cDNA进行改造以提高scFv的产量。这些改造包括：（i）将烟草病程相关蛋白PR1a的信号肽（SP）与scFv cDNA的N末端框内添加；（ii）来自烟草蚀纹病毒的5'-非翻译区（TEV前导序列），与SP的N末端融合；（iii）内质网保留信号肽KDEL，添加到scFv蛋白的C末端。使用一种涉及果胶酶的改良破坏方法，当植物前导序列、TEV序列和KDEL肽都存在于表达构建体中时，总scFv的最高表达量（344 ng scFv/g细胞）出现。虽然添加KDEL序列显著提高了蛋白质总产量5.4倍，但并没有增加分泌蛋白的总量。这些研究表明，虽然SP在促进scFv的分泌方面非常重要，但即使TEV和KDEL序列都显著提高了总蛋白水平，它对提高scFv分泌水平的影响也很小。
