A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins
A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins
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DOI：
10.1016/j.jbiotec.2006.06.018
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发表时间：
2006-12-15
影响因子：
4.1
通讯作者：
Imataka, Hiroaki
中科院分区：
文献类型：
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作者：
Mikami, Satoshi;Kobayashi, Tominari;Imataka, Hiroaki
由于大量的真核蛋白是糖蛋白，因此需要一种有效且容易获得的用于生产重组糖蛋白的无细胞系统。我们已经成功地开发了一个有效的无细胞翻译系统，来自单克隆抗体生产杂交瘤用于此目的。虽然HeLa细胞的提取物对于产生N-糖基化形式的人类免疫缺陷病毒1型包膜蛋白120（gp 120）是非常低效的，但是杂交瘤提取物能够完全N-糖基化gp 120。在无细胞翻译期间，观察到杂交瘤提取物中的e1 F2 α和eIF 2 α-激酶由于必需补充物磷酸肌酸和ATR的存在而变得磷酸化。向提取物中添加重组GADD 34和/或K3 L有效地降低了eIF 2 α的磷酸化，从而增加了蛋白质合成。利用这种改进的系统，成功地合成了具有生物活性的人绒毛膜促性腺激素（hCG），一种由α和β亚基组成的糖蛋白激素。总之，添加GADD 34/K3 L的杂交瘤提取物应该成为生产重组糖蛋白的有用工具。(c)2006 Elsevier B. V.保留所有权利。
Since a large number of eukaryotic proteins are glycoproteins, an efficient and easily available cell-free system for the production of recombinant glycoproteins is needed. We have successfully developed an efficient cell-free translation system derived from a monoclonal antibody-producing hybridoma for this purpose. While extracts from HeLa cells were very inefficient for production of an N-glycosylated form of human immunodeficiency virus type-1 envelope protein 120 (gp120), the hybridoma extract was able to fully N-glycosylate gp120. During cell-free translation, e1F2 alpha and eIF2 alpha-kinases in the hybridoma extracts were observed to become phosphorylated due to the presence of essential supplements creatine phosphate and ATR Addition of recombinant GADD34 and/or K3L to the extract efficiently lowered the phosphorylation of eIK alpha, and thereby increased protein synthesis. By using this improved system, biologically active human choriogonadotropin (hCG), a glycoprotein hormone consisting of alpha and beta subunits was successfully synthesized. In conclusion, the hybridoma extract supplemented with GADD34/K3L should become a useful tool to produce recombinant glycoproteins. (c) 2006 Elsevier B.V. All rights reserved.