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        Incomplete Polypeptides of In Vitro Translation for Epitope Localization

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        In vitro protein synthesis often leads to the production of polypeptide chains of intermediate lengths in addition to the full-size polypeptide. This accumulation of nascent chains of discrete sizes has been observed for a variety of proteins and has been attributed mainly to ribosome pausing at specific sites of mRNA owing to rare codons (1 ,2 ) or to the secondary structure of the template mRNA (3 ). Recently, Tokatlidis et al. have presented evidence that intermediate-length polypeptide chains obtained during cell-free translation of the β-subunit of Escherichia coli tryptophan synthase do not correspond to temporary pauses, but rather to incomplete N-terminal chains owing to mRNA cleavage at preferential sites (4 ). Whatever the reason leading to the discrete pattern of N-terminal nascent chains observed in cell-free transcription-translation systems, we have taken advantage of this situation to determine, during the in vitro gene expression of the β-subunit of tryptophan synthase, the size of the smallest N-terminal fragments recognized by monoclonal antibodies (MAbs) directed against this protein (5 ). Since this method permits delineation of the C-terminal border of the epitope, it could be useful to apply this approach to identify an appropriate region for further epitope mapping by other methods, such as those using synthetic peptides. In addition to the discrete pattern generated spontaneously during gene expression, truncated protein synthesis can be obtained either by arrest of mRNA translation at predetermined sites by oligonucleotides complementary to defined coding sequences within the mRNA (6 ), by using DNA fragments generated with enzymes (7 ), or by PCR (8 ,9 ).
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