We have introduced long precursor peptides directly into the endoplasmic reticulum (ER) of a mutant cell line (T2-Db) that lacks the ability to transport peptides from the cytosol to the ER in a transporter associated with antigen processing (TAP) dependent way. This was done by expressing various influenza A-derived peptides containing the naturally processed epitope ASNENMDAM (366-374) preceded by the influenza hemagglutinin ER translocation sequence. Peptides derived from these minigenes that became associated with Db were isolated and identified by combined reversed phase liquid chromatography and detection by cytotoxic T lymphocytes. Our results establish that NH2-terminal extensions of at least 40 residues can be trimmed from peptides entering the ER, but that proteolysis of larger proteins may be limited.

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