Attallah Carolina, Etcheverrigaray Marina, Kratje Ricardo, Oggero Marcos
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo" - C.C. 242, S3000ZAA, Santa Fe, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo" - C.C. 242, S3000ZAA, Santa Fe, Argentina.
Protein Expr Purif. 2017 Apr;132:27-33. doi: 10.1016/j.pep.2017.01.003. Epub 2017 Jan 10.
Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.
信号肽（SPs）是重组蛋白生产中的关键元件；然而，除了中国仓鼠卵巢细胞（CHO）外，关于哺乳动物细胞中不同信号肽的信息很少。为了研究不同信号肽引导绿色荧光蛋白（GFP）和单链抗体-融合蛋白（scFv-Fc）沿分泌途径运输的效率；以瞬时和稳定的方式转染了CHO-K1、人胚肾293（HEK293）和NS0细胞系。评估了人天青杀素（AZ）、修饰的人白蛋白（mSA）、修饰的灰仓鼠免疫球蛋白κ链V III区MOPC 63样（mIgκ C）和修饰的人免疫球蛋白κ链V III区VG（mIgκ H）的信号肽。通过荧光显微镜和流式细胞术对分泌途径内的GFP评估信号肽将前肽转运穿过内质网（ER）膜的效率，通过抗原特异性间接酶联免疫吸附测定（ELISA）对细胞外的scFv-Fc进行评估。无论转基因拷贝数如何，mSA信号肽均成功地引导了这些不同类型哺乳动物细胞中活性蛋白的分泌。这项工作的目的是证明修饰后的SA信号肽可用于使用相同表达载体的不同哺乳动物细胞。
