Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences

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

Overexpression of full-length core protein of hepatitis C virus by Escherichia coli cultivated in stirred tank fermentor

The mature core protein of the Hepatitis C virus [HCVC173] carrying pelB as a signal peptide [PelB::core] was overexpressed in Escherichia coli as 18% and 23.3% of the host's total protein, in flask and fermentor cultivation, respectively. A final specific yield of 25 +/- 1 mg HCVC173/g dry cell weight and an overall productivity of 51 +/- 1 mg HCVC173/l/h were obtained in the stirred-tank fermentor. The recombinant PelB::core protein was overexpressed as the inclusion body [IB] form, higher than the expected level when compared to the HCVC173, which was also showed by the analysis of secondary structure of mRNAs and calculation of the Codon Adaptation Index of the gene. The results showed that the combined effects of protein fusion and the signal sequence significantly enhanced the production of recombinant mature HCVC173 in E. coli. Therefore, the fusion form of the mature HCV core protein and the conditions defined in this study provide an alternative strategy for HCVC173 production in high cell density culture of E. coli