Insertion of the designed helical linker led to increased expression of tf-based fusion proteins.
Insertion of the designed helical linker led to increased expression of tf-based fusion proteins.
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DOI：
10.1007/s11095-008-9767-0
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发表时间：
2009-03
影响因子：
3.7
通讯作者：
Shen, Wei-Chiang
中科院分区：
文献类型：
--
作者：
Amet, Nurmamet;Lee, Hsin-Fang;Shen, Wei-Chiang
通过在两个蛋白质结构域之间插入螺旋接头，证明转铁蛋白（Tf）融合蛋白的高水平表达。基于转铁蛋白的融合蛋白被设计成含有编码插入蛋白质结构域之间的螺旋接头的寡核苷酸。将质粒构建体转染到HEK 293细胞中，并从条件无血清培养基中纯化分泌的融合蛋白。使用抗hGH、G-CSF或Tf抗体，使用SDS-PAGE和蛋白质印迹评估表达;使用Quantity One软件分析蛋白条带。在Nb 2细胞增殖试验中评估了由人生长激素（hGH）和Tf组成的融合蛋白的功能。含有螺旋接头的融合蛋白hGH-（H4）2-Tf和Tf-（H4）2-hGH的表达分别比不含螺旋接头的hGH-Tf融合蛋白高1.7倍和2.4倍，ED 50低2倍。表达量为Tf-G-CSF融合蛋白的11.2倍。螺旋接头的Tf融合蛋白的引入导致了高水平的表达，提高了体外生物活性。这种方法提供了一种简单的方法来增加其他融合蛋白的低表达。
To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains. Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of human growth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays. The fusion proteins containing a helical linker, hGH-(H4)2-Tf and Tf-(H4)2-hGH, were expressed 1.7- and 2.4-fold higher, respectively, with a 2-fold lower ED50 than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)2-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein. The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.
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DOI：
10.1093/protein/14.8.529
发表时间：
2001-08-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Arai, R;Ueda, H;Nagamune, T
通讯作者：
Nagamune, T
影响因子：
5.8
作者：
Ishikawa, M;Nimura, A;Tanaka, T
通讯作者：
Tanaka, T
影响因子：
4.5
作者：
Park, E;Starzyk, RM;Putney, SD
通讯作者：
Putney, SD
DOI：
10.1073/pnas.95.11.5929
发表时间：
1998-05-26
影响因子：
11.1
作者：
Robinson, CR;Sauer, RT
通讯作者：
Sauer, RT
影响因子：
3.8
作者：
Kavoosi, Mojgan;Creagh, A. Louise;Haynes, Charles A.
通讯作者：
Haynes, Charles A.