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Expression and -N end rule

Expression and -N end rule - Protocols and Methods Forum

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Old 04-29-2009, 12:26 PM
Alejandro Miguel Martin Dunn
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Default Expression and -N end rule



Shifali,

the N-end rule works at the cytoplasm; it has no influence on the
half-life of proteins secreted into the ER (as should be the case for
your venom protein in Pichia). Also (at least in E. coli) remember that
the amino-terminal methionine is not always removed by MAP; this depends
on the bulkiness of the side chain for the second a.a. Unsurprisingly,
residues which are destabilizing according to the N-end rule often
inhibit removal of the N-terminal Met by MAP when they are in the second
position.

how did you check for expression in yeast? did you also test cell
lysates? Straight SDS-PAGE or Western blotting?


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