Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris
The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme’s capability, thus simplify...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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The Genetics Society of Japan
2013
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| Online Access: | http://psasir.upm.edu.my/id/eprint/28061/1/28061.pdf |
| _version_ | 1825947242537680896 |
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| author | Ahmad Latiffi, Amaliawati Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Oslan, Siti Nurbaya Basri, Mahiran |
| author_facet | Ahmad Latiffi, Amaliawati Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Oslan, Siti Nurbaya Basri, Mahiran |
| author_sort | Ahmad Latiffi, Amaliawati |
| collection | UPM |
| description | The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme’s capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa. |
| first_indexed | 2024-03-06T08:10:14Z |
| format | Article |
| id | upm.eprints-28061 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T08:10:14Z |
| publishDate | 2013 |
| publisher | The Genetics Society of Japan |
| record_format | dspace |
| spelling | upm.eprints-280612016-09-26T03:54:14Z http://psasir.upm.edu.my/id/eprint/28061/ Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris Ahmad Latiffi, Amaliawati Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Oslan, Siti Nurbaya Basri, Mahiran The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme’s capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa. The Genetics Society of Japan 2013 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/28061/1/28061.pdf Ahmad Latiffi, Amaliawati and Salleh, Abu Bakar and Raja Abdul Rahman, Raja Noor Zaliha and Oslan, Siti Nurbaya and Basri, Mahiran (2013) Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris. Genes & Genetic Systems, 88 (2). pp. 85-91. ISSN 1341-7568; ESSN: 1880-5779 https://www.jstage.jst.go.jp/article/ggs/88/2/88_85/_article 10.1266/ggs.88.85 |
| spellingShingle | Ahmad Latiffi, Amaliawati Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Oslan, Siti Nurbaya Basri, Mahiran Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris |
| title | Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris |
| title_full | Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris |
| title_fullStr | Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris |
| title_full_unstemmed | Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris |
| title_short | Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris |
| title_sort | secretory expression of thermostable alkaline protease from bacillus stearothermophilus fi by using native signal peptide and α factor secretion signal in pichia pastoris |
| url | http://psasir.upm.edu.my/id/eprint/28061/1/28061.pdf |
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