Au L C, Yang F Y, Yang W J, Lo S H, Kao C F
Department of Medical Research, Veterans General Hospital-Taipei, Taiwan, Republic of China.
Biochem Biophys Res Commun. 1998 Jul 9;248(1):200-3. doi: 10.1006/bbrc.1998.8929.
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 6 x 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.
合成基因在基因工程和蛋白质工程中非常有用。在此，我们提出一种构建合成基因的通用方法。短寡核苷酸在高严格条件下通过连接酶链反应（LCR）连接以产生“单位片段”，然后通过聚合酶链反应将这些片段融合以形成全长基因序列。该方法简单且准确，对序列和长度没有限制。在本报告中，根据大肠杆菌的密码子偏好合成了重组瘦素基因。此外，在合成基因中，将第54位唯一的甲硫氨酸替换为亮氨酸，并在N端添加了一个甲硫氨酸。该基因被克隆到pQE-31表达载体中并在大肠杆菌中表达。通过镍-亚氨基二乙酸（Ni-NTA）亲和柱产生并纯化了大量含有6×组氨酸标签的重组瘦素。最后，通过在甲硫氨酸残基处用溴化氰（CNBr）切割去除标签后，释放出完整的瘦素-L54。
