TY - JOUR ID - 7164 TI - Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences JO - Iranian Journal of Biotechnology JA - IJB LA - en SN - 1728-3043 AU - Karkhane, Ali Asghar AU - Yakhchali, Bagher AU - Rastgar Jazii, Ferdous AU - Hemmat, Jafar AU - Shariati, Parvin AU - Khodabandeh, Mahvash AU - Zomorodipoor, Alireza AD - Department of Industrial and Environmental Biotechnology at the National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran. AD - Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST),P.O. Box 15815-3538 Y1 - 2012 PY - 2012 VL - 10 IS - 4 SP - 255 EP - 262 KW - Bacillus thermocatenulatus Lipase KW - capillary isoelectric focusing KW - Inclusion bodies KW - Periplasmic space KW - Tandem mass spectrometry DO - N2 - Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful andmost of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role ofnative 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 withthe 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. UR - https://www.ijbiotech.com/article_7164.html L1 - https://www.ijbiotech.com/article_7164_cf7cdb23a04a72c97a1c08fa099988aa.pdf ER -