Escherichia coli SIGNAL PEPTIDASES RECOGNIZE AND CLEAVE THE SIGNAL SEQUENCE OF а-AMYLASE FROM Bacillus licheniformis
The gene encoding the а-amylase from Bacillus licheniformis was cloned, with and without the native signal sequence, and expressed in Escherichia coli, resulting in the production of the recombinant protein in the cytoplasm as insoluble but enzymatically active aggregates. Expression with low concentration of the inducer at low temperature resulted in the production of the recombinant protein in soluble form in a significant amount. The protein produced with signal sequence was exported to the extracellular medium, whereas there was no export of the protein produced from the gene without the signal sequence. Similarly, the а-amylase activity in the culture medium increased with time after induction for the protein produced with signal sequence. Molecular mass determinations by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing of the purified recombinant б-amylase from the extracellular medium revealed that the native signal peptide was cleaved by the E. coli signal peptidases between Ala <sup>28</sup> and Ala <sup>29</sup>. It seems possible that the signal peptide of а-amylase from B. licheniformis can be used for the secretion of other recombinant proteins produced using a E. coli expression system.