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Karkhane, A., Yakhchali, B., Rastgar Jazii, F., Hemmat, J., Shariati, P., Khodabandeh, M., Zomorodipoor, A. (2012). Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences. Iranian Journal of Biotechnology, 10(4), 255-262.
Ali Asghar Karkhane; Bagher Yakhchali; Ferdous Rastgar Jazii; Jafar Hemmat; Parvin Shariati; Mahvash Khodabandeh; Alireza Zomorodipoor. "Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences". Iranian Journal of Biotechnology, 10, 4, 2012, 255-262.
Karkhane, A., Yakhchali, B., Rastgar Jazii, F., Hemmat, J., Shariati, P., Khodabandeh, M., Zomorodipoor, A. (2012). 'Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences', Iranian Journal of Biotechnology, 10(4), pp. 255-262.
Karkhane, A., Yakhchali, B., Rastgar Jazii, F., Hemmat, J., Shariati, P., Khodabandeh, M., Zomorodipoor, A. Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences. Iranian Journal of Biotechnology, 2012; 10(4): 255-262.

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

Article 4, Volume 10, Issue 4, Autumn 2012, Page 255-262  XML PDF (588.8 K)
Authors
Ali Asghar Karkhane email 1; Bagher Yakhchali1; Ferdous Rastgar Jazii1; Jafar Hemmat2; Parvin Shariati1; Mahvash Khodabandeh1; Alireza Zomorodipoor1
1Department of Industrial and Environmental Biotechnology at the National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran.
2Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST),P.O. Box 15815-3538
Abstract
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.
Keywords
Bacillus thermocatenulatus Lipase; capillary isoelectric focusing; Inclusion bodies; Periplasmic space; Tandem mass spectrometry
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