Gene synthesis by a LCR-based approach:: High-level production of leptin-L54 using synthetic gene in Escherichia coli
Gene synthesis by a LCR-based approach:: High-level production of leptin-L54 using synthetic gene in Escherichia coli
复制标题
DOI：
10.1006/bbrc.1998.8929
复制
发表时间：
1998-07-09
影响因子：
3.1
通讯作者：
Kao, CF
中科院分区：
文献类型：
--
作者：
Au, LC;Yang, FY;Kao, CF
人工合成基因在基因工程和蛋白质工程中非常有用。在这里，我们提出了一个通用的方法来构建合成基因。短的寡核苷酸通过连接酶链式反应（LCR）在高严格条件下连接以制备“单元片段”，然后通过聚合酶链式反应融合以形成全长基因序列。该方法简单、准确，对序列和长度没有限制。本研究根据大肠杆菌的密码子偏好性，合成了重组瘦素基因。此外，在合成基因中引入了54位的唯一Met替换为Leu和在N-末端添加Met。将该基因克隆到pQE-31表达载体中，并在E.杆菌利用Ni-NTA亲和层析柱纯化了含有6 × His标签的重组瘦素。最后，在通过在Met残基处的CNBr切割去除标签后，释放完整的瘦素-L54。(C)北京：科学出版社.
Synthetic genes are very useful in genetic and protein engineering. Here we propose a general method for construction of synthetic genes. Short oligonucleotides are joined through ligase chain reaction (LCR) in high stringency conditions to make "unit fragments" which are then fused to form a full-length gene sequence by polymerase chain reaction. The procedure is simple and accurate and does not place constraints on sequence and length. In this report, a recombinant leptin gene was synthesized according to the codon preference of Escherichia coli. Besides, a substitution of the only Met at position 54 for Leu and an addition of a Met at the N-terminus were introduced in the synthetic gene. The gene was cloned in the pQE-31 expression vector and was expressed in E. coli. A large amount of recombinant leptin containing 6x His tag was produced and purified by Ni-NTA affinity column. Finally, intact leptin-L54 was released after removing the tag by CNBr cleavage at the Met residue. (C) 1998 Academic Press.