Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli
Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli
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DOI：
10.1016/j.pep.2004.02.005
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发表时间：
2004-06-01
影响因子：
1.6
通讯作者：
Nascimento, ALTO
中科院分区：
文献类型：
--
作者：
Neves, FO;Ho, PL;Nascimento, ALTO
干扰素(IFN)是脊椎动物重要的防御机制。在这项工作中，我们描述了使用聚合酶链式反应作为一种一步合成DNA序列的方法的基因合成和组装。设计的寡核苷酸基于大肠杆菌密码子的使用，合成了两个干扰素基因：一个含有已知的DNA序列，另一个是突变形式，其中两个半胱氨酸氨基酸残基被丝氨酸取代，以提高蛋白质的稳定性。将DNA序列克隆到PAE中，这是一种允许在有或没有组氨酸标签的情况下表达异源蛋白的大肠杆菌载体。用抗人干扰素多克隆抗体通过Western blotting和ELISA法鉴定重组人干扰素。重组His标记蛋白经Ni2+离子交换柱层析一步纯化，无His标记蛋白通过包涵体广泛洗涤得到纯化，最终产量分别为210 mg/L和75 mg/His。抗病毒实验表明，在该系统中表达的重组人干扰素具有生物活性(类似于1，1×10(8)IU/mg)。这里描述的组合方法被证明是具有成本效益的，并可以扩展到其他感兴趣的基因/蛋白质。(C)2004 Elsevier Inc.保留所有权利。
Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni2+-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being similar to210 and 75 mg/L, respectively. The rhIFNs expressed within this system were biologically active (similar to1,1 x 10(8) IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest. (C) 2004 Elsevier Inc. All rights reserved.