Academic Journal
High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides
| العنوان: | High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides |
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| المؤلفون: | Markian S. Bahniuk, Abdullah K. Alshememry, Larry D. Unsworth |
| المصدر: | BioTechniques, Vol 61, Iss 6, Pp 297-304 (2016) |
| بيانات النشر: | Taylor & Francis Group, 2016. |
| سنة النشر: | 2016 |
| المجموعة: | LCC:Biology (General) |
| مصطلحات موضوعية: | elastin-like polypeptides, recursive directional ligation, inverse temperature transitions, concatemerization, denaturing metal affinity chromatography, marginally soluble protein purification, Biology (General), QH301-705.5 |
| الوصف: | The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250–660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1940-9818 0736-6205 |
| Relation: | https://doaj.org/toc/0736-6205; https://doaj.org/toc/1940-9818 |
| DOI: | 10.2144/000114482 |
| URL الوصول: | https://doaj.org/article/2649bd0c8afb45648e2c76ec1c392116 |
| رقم الانضمام: | edsdoj.2649bd0c8afb45648e2c76ec1c392116 |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 19409818 07366205 |
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| DOI: | 10.2144/000114482 |