Description:
<jats:p>Two genetically engineered variants of the <jats:italic>Bacillus licheniformis</jats:italic>β‐lactamase gene were expressed in <jats:italic>Escherichia coli</jats:italic>. One variant coded for the <jats:italic>exo‐small</jats:italic> mature enzyme without the signal peptide. The other coded for the <jats:italic>exo‐large</jats:italic> mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme‐EDTA treatment, the signal‐less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N‐terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal‐less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding <jats:italic>in vitro</jats:italic> of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large Δ<jats:italic>G</jats:italic> of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non‐native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these <jats:italic>B. licheniformis</jats:italic> lactamases fold much faster than <jats:italic>E. coli</jats:italic>β‐lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.</jats:p>