Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli.
The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.
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Amgen Inc., Amgen Center, Thousand Oaks, CA 91320, USA.
Rohde
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Thomason
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Kenney
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WC
Lu
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018388:Codon, Terminator; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014364:Tryptophan / Q000235:genetics*
D018388:Codon, Terminator; D004274:DNA, Recombinant; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D014364:Tryptophan