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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418719/ https://www.ncbi.nlm.nih.gov/pubmed/30965969 http://dx.doi.org/10.3390/polym9120669 |
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author | Wu, Yue Kang, Zhao Tian, Zhifang Wu, Mingyang Wang, Jiannan |
author_facet | Wu, Yue Kang, Zhao Tian, Zhifang Wu, Mingyang Wang, Jiannan |
author_sort | Wu, Yue |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-6418719 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-64187192019-04-02 Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion Wu, Yue Kang, Zhao Tian, Zhifang Wu, Mingyang Wang, Jiannan Polymers (Basel) Article 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. MDPI 2017-12-03 /pmc/articles/PMC6418719/ /pubmed/30965969 http://dx.doi.org/10.3390/polym9120669 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Wu, Yue Kang, Zhao Tian, Zhifang Wu, Mingyang Wang, Jiannan Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion |
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 | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418719/ https://www.ncbi.nlm.nih.gov/pubmed/30965969 http://dx.doi.org/10.3390/polym9120669 |
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