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.
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.
Kerrigan
John J
JJ
Department of Biological Reagents and Assay Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA. john.j.kerrigan@gsk.com
McNulty
Dean E
DE
Burns
Matthew
M
Allen
Kimberly E
KE
Tang
Xiaoyan
X
Lu
Quinn
Q
Trulli
Janice M
JM
Johanson
Kyung O
KO
Kane
James F
JF
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D016368:Frameshift Mutation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D012357:RNA, Transfer, Lys / Q000235:genetics*; D012065:Relaxin / Q000235:genetics* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D003062:Codon; D012357:RNA, Transfer, Lys; D012065:Relaxin; D001120:Arginine