2. Right - growing to a higher OD before induction and reducing the induction duration may improve expression of toxic proteins. However, the Origami strain yields only insoluble protein even after a relatively short period of induction (2hrs). Thus, choosing a different bacterial strain is a good idea.
3. Wrong - apparently, growth of cells that start expressing NS4B is arrested. A richer medium may enhance protein expression (if the medium is buffered), provided the expressed protein does not induce any toxic effects.
4. Right - export of potentially toxic proteins to the periplasm, may render them less toxic. However, in order to avoid aggregates formation, the synthesis of the expressed protein should be slowed to suit the rate of the transport systems. Aggregates can still form in the periplasm, if the protein contains hydrophobic segments, as is the case with NS4B.
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