Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion
In order to investigate the impacts on the structure and biomedical function of typical fragments derived from repetitive and non-repetitive regions of the Bombyx mori silk fibroin heavy chain, several block combination genes (gs16f1, gs16f4, gs16f8, and gs16f12) were designed, cloned into a fusion...
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| Format: | Article |
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MDPI AG
2017-12-01
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| Series: | Polymers |
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| Online Access: | https://www.mdpi.com/2073-4360/9/12/669 |
| _version_ | 1811216116419133440 |
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| author | Yue Wu Zhao Kang Zhifang Tian Mingyang Wu Jiannan Wang |
| author_facet | Yue Wu Zhao Kang Zhifang Tian Mingyang Wu Jiannan Wang |
| author_sort | Yue Wu |
| collection | DOAJ |
| description | In order to investigate the impacts on the structure and biomedical function of typical fragments derived from repetitive and non-repetitive regions of the Bombyx mori silk fibroin heavy chain, several block combination genes (gs16f1, gs16f4, gs16f8, and gs16f12) were designed, cloned into a fusion protein expression vector tagged with glutathione S-transferase (GST), and expressed in Escherichia coli. Fusion proteins GST-GS16F1, GST-GS16F4, and GST-GS16F8 were purified by GST affinity chromatography, and single bands were identified by SDS-PAGE. Under optimal initial cell density, in ducer concentration and induction expression time, the yield of purified GST-GS16F1, GST-GS16F4, and GST-GS16F8 per liter of bacterial culture reached 79, 53, and 28 mg, respectively. Mass spectrometry revealed molecular weights for GST-GS16F1, GST-GS16F4, and GST-GS16F8 of 37.7, 50.0, and 65.7 kDa, respectively, consistent with the theoretical values of 37.4, 49.4, and 65.5 kDa. Similarly, measured values of pI were 5.35, 4.5, and 4.2 for the fusion proteins, consistent with predicted values of 5.34, 4.44, and 4.09. CD spectra showed the molecular conformation of GS16F1 was mainly β-sheet structure, while more stable α-helix structure formed in GS16F4 and GS16F8. |
| first_indexed | 2024-04-12T06:34:02Z |
| format | Article |
| id | doaj.art-c1b2728908ad42639043f30ced3212e1 |
| institution | Directory Open Access Journal |
| issn | 2073-4360 |
| language | English |
| last_indexed | 2024-04-12T06:34:02Z |
| publishDate | 2017-12-01 |
| publisher | MDPI AG |
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| series | Polymers |
| spelling | doaj.art-c1b2728908ad42639043f30ced3212e12022-12-22T03:43:56ZengMDPI AGPolymers2073-43602017-12-0191266910.3390/polym9120669polym9120669Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk FibrionYue Wu0Zhao Kang1Zhifang Tian2Mingyang Wu3Jiannan Wang4National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu Province, ChinaNational Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu Province, ChinaNational Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu Province, ChinaNational Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu Province, ChinaNational Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu Province, ChinaIn order to investigate the impacts on the structure and biomedical function of typical fragments derived from repetitive and non-repetitive regions of the Bombyx mori silk fibroin heavy chain, several block combination genes (gs16f1, gs16f4, gs16f8, and gs16f12) were designed, cloned into a fusion protein expression vector tagged with glutathione S-transferase (GST), and expressed in Escherichia coli. Fusion proteins GST-GS16F1, GST-GS16F4, and GST-GS16F8 were purified by GST affinity chromatography, and single bands were identified by SDS-PAGE. Under optimal initial cell density, in ducer concentration and induction expression time, the yield of purified GST-GS16F1, GST-GS16F4, and GST-GS16F8 per liter of bacterial culture reached 79, 53, and 28 mg, respectively. Mass spectrometry revealed molecular weights for GST-GS16F1, GST-GS16F4, and GST-GS16F8 of 37.7, 50.0, and 65.7 kDa, respectively, consistent with the theoretical values of 37.4, 49.4, and 65.5 kDa. Similarly, measured values of pI were 5.35, 4.5, and 4.2 for the fusion proteins, consistent with predicted values of 5.34, 4.44, and 4.09. CD spectra showed the molecular conformation of GS16F1 was mainly β-sheet structure, while more stable α-helix structure formed in GS16F4 and GS16F8.https://www.mdpi.com/2073-4360/9/12/669Bombyx morisilk fibroinheavy chainprotein expressionE. colicharacterization |
| spellingShingle | Yue Wu Zhao Kang Zhifang Tian Mingyang Wu Jiannan Wang Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion Polymers Bombyx mori silk fibroin heavy chain protein expression E. coli characterization |
| title | Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion |
| title_full | Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion |
| title_fullStr | Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion |
| title_full_unstemmed | Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion |
| title_short | Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion |
| title_sort | biosynthesis and characterization of recombinant silk like polypeptides derived from the heavy chain of silk fibrion |
| topic | Bombyx mori silk fibroin heavy chain protein expression E. coli characterization |
| url | https://www.mdpi.com/2073-4360/9/12/669 |
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