Frameshift events associated with the lysyl-tRNA and the rare arginine codon, AGA, in Escherichia coli: A case study involving the human Relaxin 2 protein
- 作者：John J. Kerrigan ; Dean E. McNulty ; Matthew Burns ; Kimberly E. Allen ; Xiaoyan Tang ; Quinn Lu ; Janice M. Trulli ; Kyung O. Johanson ; James F. Kane
- 关键词：Rare codons ; Relaxin 2 ; E. coli ; pRR692 ; Bl21 (DE3) ; Frameshift events
- 刊名：Protein Expression and Purification
- 出版年：2008
- 期刊代码：184_10465928
- 类别：bio
- 出版时间：August 2008
- 卷：60
- 期：2
- 页码：110-116
- 文件大小：717 K
摘要
Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in E. coli BL21 (DE3), several forms of the protein were made. One species had the expected molecular weight (19,084 Da). A second species observed had a molecular weight of 21,244 Da. A third minor species had a molecular weight of 17,118 Da. These aberrant molecular weights can be explained as follows. First, a sequence CGA-AAA-AAG-AGA, containing the rare arginine codons CGA and AGA was the site of the +1 frameshift that generated the 21,244 Da species. Since there was a limited supply of this arginyl-tRNA, the peptidyl-tRNA moved +1 nucleotide to occupy the codon and resumed protein synthesis. Second, a −1 frameshift associated with ‘slippery A’ sequence XXA-AAA-AAG accounted for 10%of the product with a mass of 17,118 Da. Presumably, the shift to −1 also occurred because there was a paucity of the 
. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species.