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Construction of improved mammalian expression vectors

机译：改善哺乳动物表达载体的构建

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To improve the productivity of recombinant protein using mammalian cells, we modified an expression vector particularly in its post promoter region to regulate the leader sequence of mRNA and obtained improved vectors with significantly high production efficiency in this paper. Here, the original vector we used, pK2SR alpha/hTfr, for the production of a foreign protein, i.e., human transfenin in our case. Our attention was particularly paid to the translation initiation codons in the 5'-leader sequence of mRNA, which we noted varying when the original vector was used: One of the Tfr mRNAs contained additional initiator codon, ATG~3, near the 5' cap site. We constructed a vector by changing ATG~3 to ATC~3 and this modified vector turned out to showthe highest productivity among other modified vectors. Several Chinese hamster ovary (CHO) cell lines producing recombinant human transferrin (rhTfr) were established using an improved dicistronic vector, pSR gamma SIGMA-1/hTfr. Productivities of thesenew cell lines were two to three times higher than that of cell lines obtained with the pK2SR alpha/hTfr vector. Purified rhTfr exhibited reversible iron-binding capability in the manner indistinguishable from that of serum derived hTfr.
机译：为了提高使用哺乳动物细胞的重组蛋白的生产率，我们修饰了表达载体，特别是在其后启动子区域中，以调节mRNA的前导序列，并在本文中获得了具有显着高的生产效率的改善载体。这里，我们使用的原始载体，PK2SRα/ HTFR，用于生产外国蛋白质，即我们的案例中的人体变细胞。我们的注意力特别是MRNA的5'-前导序列中的翻译起始密码子，我们在使用原始载体时，我们注意到了：TFR MRNA中的一个包含额外的发起者密码子，ATG〜3，靠近5'帽附近地点。我们通过改变ATG〜3至ATC〜3来构建了矢量，并且该修改的向量证明了其他修改向量之间的高生产率。使用改进的Dicistronic载体，PSRγsigma-1 / HTFR建立了产生重组人转移素（RHTFR）的几种中国仓鼠卵巢（CHO）细胞系。表达型细胞系的产品高于PK2SRα/ HTFR向量获得的细胞系的两到三倍。纯化的rhTFR以可区分的方式表现出可逆的铁结合能力，该方法与血清衍生HTFR的方式难以区分。

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