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-ac id precursor synthesized in mammalian cells, with removal of 81 N-term inal and 51 C-terminal amino acids. In order to produce directly the m ature 109-amino acid PDGF-B peptide as a recombinant protein in Escher ichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translati on termination codon TGA by in vitro mutagenesis. Expression of this D NA via a plasmid vector in E. coli resulted in production of two disti nct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization em ploying N- and C-terminal amino acid sequencing and MS analyses indica ted 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 conta ining 160 amino acids. Characterization of a unique tryptic peptide de rived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon a t position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owi ng to the high rate of translation readthrough of TGA codons in this a nd occasionally other proteins, it appears that the use of TGA as a tr anslation termination codon for proteins to be expressed in E. coli sh ould be avoided when possible.