“Export and Folding of Signal-sequenceless B. licheniformis -lactamase in E. coli”

FRATE, M.C.; SANTOS, J; LIETZ, E.; FINK, A.L.; ROSSI, J.P.F.C; ERMACORA, MR

EUROPEAN JOURNAL OF BIOCHEMISTRY

Blackwell Pub.,

Lugar: London; Año: 2000 p. 3836 - 3847

0014-2956

Two genetically engineered variants of the Bacillus licheniformis b-lactamase gene were expressed inEscherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other codedfor the exo-large 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 theperiplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to alesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, whichmay be of importance for our understanding of cellular compartments. The fact that a signal-less protein istranslocated 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 in vitro of the two variants. Nodifferences were found between them. In the absence of denaturant, they are completely folded, fully active andhave a large DG 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 thermodynamicpartitioning between folding and export less likely. In addition, kinetic measurements indicated that theseB. licheniformis lactamases fold much faster than E. coli b-lactamase. This behavior suggests that they areexported by a kinetically controlled process, mediated by one or more still unidentified interactions that slowfolding and allow a folding intermediate to enter the export pathway.Keywords: b-lactamase; protein export; protein folding; signal peptide.