Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3).
Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins. However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility. Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E. coli and specifically to a tandem pair of AGA codons. The yield of protease can be improved by replacing these rare arginine codons with synonymous ones or by increasing the supply of cognate tRNA that is available to the cell. Furthermore, we show that when ribosomes become stalled at rare arginine codons in the TEV protease mRNA, the nascent polypeptides are targeted for proteolytic degradation in BL21(DE3) cells by a mechanism that does not involve tmRNA-mediated peptide tagging.
Kapust
Rachel B
RB
Protein Engineering Section, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA.
Routzahn
Karen M
KM
Waugh
David S
DS
D018528:ATP-Binding Cassette Transporters; D001120:Arginine / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides / Q000235:genetics; D017800:Potyvirus / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012343:RNA, Transfer / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D020080:Tandem Repeat Sequences; D018911:Trinucleotide Repeats; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C461599:maltose transport system, E coli; D012343:RNA, Transfer; D001120:Arginine; D010450:Endopeptidases; C099488:TEV protease