Gene synthesis by a LCR-based approach:: High-level production of leptin-L54 using synthetic gene in Escherichia coli

被引:58
作者
Au, LC [1 ]
Yang, FY [1 ]
Yang, WJ [1 ]
Lo, SH [1 ]
Kao, CF [1 ]
机构
[1] Vet Gen Hosp, Dept Med Res, Taipei 11217, Taiwan
关键词
oligonucleotide; synthetic gene; LCR; CNBr cleavage;
D O I
10.1006/bbrc.1998.8929
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:200 / 203
页数:4
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