Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful and
most of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role of
native and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli,
the lipase gene (btl2) was cloned downstream of the native Bacillus signal peptide and also in fusion with
the pelB, ansB and ansB/asp signal peptides. For this purpose, four recombinant expression vectors (pYRKP.P, pYRKP.N, pYRKP. A and pYRKP.AA) were constructed and expressed in E. coli. Osmotic shock analysis showed that recombinant lipase was overexpressed as inclusion bodies in E. coli. The lipase inclusion bodies were subsequently solublized, refolded and purified using single column ion-exchange chromatography. To evaluate localization of lipase in the cell, the purified lipases were subjected to capillary isoelectric focusing and tandem mass spectrometry. Results showed that all signal peptides were able to direct the lipase from the cytoplasm into the periplasmic space of E. coli, because the periplasmic space of E. coli is not suitable for lipase folding, the translocated lipase aggregates in this space as inclusion bodies.