Formation of disulfide bonds
Some vectors provide a N-terminal fusion of the ompA signal peptide which mediates the secretion of the recombinant protein to the periplasmic space of E. coli. There, the signal peptide is selectively cleaved by the E. coli signal peptidase. The secretion strategy is essential for the functional production of proteins containing structural disulfide bonds that are often present in naturally secreted proteins. The reducing conditions in the cytoplasm of E. coli prevent disulfide bond formation which can lead to aggregation or degradation of unfolded polypeptides.
Periplasmic secretion as a first purification step
Furthermore, periplasmic secretion separates the recombinant protein from cytosolic proteases. Since the E. coli outer membrane can be selectively degraded by mild treatment (EDTA, lysozyme etc.) the spheroplasts containing the cytosolic components can be easily removed by centrifugation.
Addition of active substances
In addition, the periplasmic space is accessible to molecules < 600 Da allowing to influence folding or stability of the recombinant protein during expression by adding active substances to the culture media (e.g. redox components, non-metabolizable sugars, ligands of the recombinant protein etc.).
Cytoplasmic or periplasmic expression
As long as a cytoplasmic recombinant protein does not include stop-transfer sequences, the advantages of periplasmic secretion are also amenable to this type of protein. However, because stop-transfer sequences are difficult to predict, it is advisable to try both strategies in parallel. Using the vectors for N-terminal Strep-tag II or 6xHistidine-tag fusion, the change from cytoplasmic to periplasmic expression (and vice versa) can be achieved by a simple cloning step via the NheI/BstBI and the EcoRV/HindIII restriction sites on the 5'- and 3'- end, respectively (see Multiple Cloning Sites).
Do you need an authentic protein?
For special applications requiring an authentic protein, several vectors encoding the factor Xa protease cleavage site adjacent to Strep-tag II/6xHistidine-tag are available allowing the complete removal of the tag. In addition to factor Xa protease cleavage sites, we offer vectors with thrombin and enterokinase cleavage
sites. For an overview of all vectors click here. Please note, that in most cases it is superfluous to remove the tag.
| Factor Xa processing |
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Protein gel demonstrating the treatment of the 15 kDa selenoprotein (carrying a N-terminal Strep-tag) with factor Xa (1:1000, w/w) at 22°C. The gene encoding the protein had been cloned into pASK-IBA6, which contains a Xa cleavage site adjacent to Strep-tag II (see Multiple Cloning Sites ).
Lane 1, protein before Xa processing; lane 2, after 1 hour incubation; lane 3, after 2 hours; lane 4, after 4 hours; lane 5, after 16 hours: homogenous authentic protein without Strep-tag II. |
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