Amino acid misincorporation during high-level expression of mouse epidermal growth factor in Escherichia coli.
To determine whether the high-level expression of foreign proteins in Escherichia coli can lead to frequent translational errors, we analyzed amino acid misincorporation in mouse epidermal growth factor (mEGF) produced as a TrpE fusion protein. The mEGF DNA does not encode phenylalanine and determining the phenylalanine content of the purified protein will measure missense errors. Using this approach, we found an error frequency of about 1 in 40 for codons differing by a single base from those for phenylalanine. This is at least ten times higher than the error rate found for normal E. coli protein synthesis and may be due to limiting supply of charged tRNAs and GTP, brought about by the high-level production of the heterologous protein. The unexpectedly high error rate has implications for the clinical use of E. coli-derived therapeutic proteins.
Scorer
C A
CA
Genetics Division, National Institute for Medical Research, London, UK.
Carrier
M J
MJ
Rosenberger
R F
RF
D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D003062:Codon / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D007525:Isoelectric Focusing; D007930:Leucine / Q000032:analysis; D051379:Mice; D010649:Phenylalanine / Q000032:analysis / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D000596:Amino Acids; D003062:Codon; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D004815:Epidermal Growth Factor; D007930:Leucine