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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.

Abstract：

: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 arginyl-tRNAArgucu. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Kerrigan JJ,McNulty DE,Burns M,Allen KE,Tang X,Lu Q,Trulli JM,Johanson KO,Kane JF

doi

10.1016/j.pep.2008.02.016

subject

Has Abstract

pub_date

2008-08-01 00:00:00

pages

110-6

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(08)00061-2

journal_volume

60

pub_type

杂志文章

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