Efficient high level expression of peptides and proteins as fusion proteins with the N-terminal domain of L9: Application to the villin headpiece helical subdomain
Efficient high level expression of peptides and proteins as fusion proteins with the N-terminal domain of L9: Application to the villin headpiece helical subdomain
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DOI：
10.1016/j.pep.2005.10.023
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发表时间：
2006-05-01
影响因子：
1.6
通讯作者：
Cho, Jae-Hyun
中科院分区：
文献类型：
--
作者：
Bi, Yuan;Tang, Yuefeng;Cho, Jae-Hyun
小到中等大小的多肽和小的边缘稳定的蛋白质的有效表达可能是困难的。开发了一种新的蛋白质融合系统以允许肽和小蛋白的表达。目的多肽通过因子Xa切割序列连接至核糖体蛋白L9（NTL 9）的N-末端结构域的C-末端。NTL 9是一种小的（56个残基）碱性蛋白。蛋白质的C-末端是α-螺旋的一部分，其延伸远离球状结构，因此可以融合另外的结构域而不改变NTL 9的折叠。NTL 9以高水平表达，可溶性极高，并且在宽的温度和pH范围内保持完全折叠。该蛋白质具有高净正电荷，有利于通过离子交换层析纯化融合蛋白。NTL 9融合物也可以通过反相HPLC容易地纯化。作为一个测试案例，我们证明了一个小的，36个残基，三螺旋束，绒毛头部亚域的高水平表达。该蛋白被广泛用作折叠研究的模型系统，并且开发简单的表达系统应有助于该亚结构域的实验研究。纯化的融合蛋白产量为70 mg/L培养物，纯化的绒毛头部亚结构域产量为24 mg/L培养物。我们还证明了使用的融合系统来表达一个较小的边缘折叠的绒毛蛋白头片段结构域的肽片段。(c)2005年爱思唯尔公司All rights reserved.
The efficient expression of small to midsize polypeptides and small marginally stable proteins can be difficult. A new protein fusion system is developed to allow the expression of peptides and small proteins. The polypeptide of interest is linked via a Factor Xa cleavage sequence to the C-terminus of the N-terminal domain of the ribosomal protein L9 (NTL9). NTL9 is a small (56 residue) basic protein. The C-terminus of the protein is part of an a-helix which extends away from the globular structure thus additional domains can be fused without altering the fold of NTL9. NTL9 expresses at high levels, is extremely soluble, and remains fully folded over a wide temperature and pH range. The protein has a high net positive charge, facilitating purification of fusion proteins by ion exchange chromatography. NTL9 fusions can also be easily purified by reverse phase HPLC. As a test case we demonstrate the high level expression of a small, 36 residue, three helix bundle, the villin headpiece subdomain. This protein is widely used as a model system for folding studies and the development of a simple expression system should facilitate experimental studies of the subdomain. The yield of purified fusion protein is 70 mg/L of culture and the yield of purified villin headpiece subdomain is 24 mg/L of culture. We also demonstrate the use of the fusion system to express a smaller marginally folded peptide fragment of the villin headpiece domain. (c) 2005 Elsevier Inc. All rights reserved.