Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli |
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| Institution: | 1. Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia;2. Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia;3. School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia;4. Malaysia Genome Institute, Ministry of Science, Technology and Innovation Malaysia, Jalan Bangi, 43000 Kajang, Selangor, Malaysia;1. Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul 02841, Republic of Korea;2. Department of Public Health Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul 02841, Republic of Korea;3. Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, 03760, Republic of Korea;4. School of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea;1. Institute of Resource Biology and Biotechnology, College of Life Science & Technology, Huazhong University of Science & Technology, Wuhan 430074, China;2. College of Life Science, South-central University for Nationalities, Wuhan 430074, China;3. Shanghai Information Center for Life Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China;4. Key Laboratory of Fermentation Engineering, Hubei University of Technology, Wuhan 430068, China;5. Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, 00076 Aalto, Finland;1. Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing, 100083 China;2. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083 China;1. Department of Pharmacy, State University of Maringá (UEM), Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil;2. Department of Physics, State University of Maringá (UEM), Av. Colombo, 5790, 87020-900, Maringá, PR, Brazil;1. College of Veterinary Medicine, Northeast Agricultural University, Harbin, PR China;2. Northeastern Science Inspection Station, China Ministry of Agriculture Key Laboratory of Animal Pathogen Biology, PR China;1. Department of Chemical Engineering, University of Waterloo, 200 University Ave West, Waterloo, ON N2L 3G1, Canada;2. Trojan Technologies, London, Ontario, Canada |
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| Abstract: | Secretion of recombinant proteins in heterologous host has drawn attention for its simpler purification and downstream processes. Searching for secretion aid molecules to improve protein secretion can be done through synthetic biology, screening of genome data and proteome-based approach. In the present study, the extracellular proteome on starch-containing medium of Bacillus lehensis G1 was analyzed to identify naturally secreted proteins with signal peptide. A total of 87 protein spots were identified by mass spectrometry, which were categorized mostly in the metabolism of carbohydrates and related molecules (20%). Over-expression and secretion studies were performed for all the 14 selected signal peptides fused to a reporter protein, cyclomaltodextrin glucanotransferase (CGTase). All clones were found to allow CGTase to be excreted into the medium, as observed and measured from the iodine plate assay and enzyme activity assay. Compared to native signal peptide (G1) of CGTase, signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ～1.7 fold the amount of β-galactosidase (cell lysis) in the medium. GAP has the highest secretion rate of 45.6 U/ml/h among all clones, where physicochemical characteristics of signal peptide play significant role. |
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| Keywords: | Proteome Signal peptide Protein secretion |
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