Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization
| Autor: | Yasuhiro Kumaki, Satoshi Tomisawa, Masakatsu Kamiya, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Tokiyoshi Ayabe, Kiminori Nakamura, Yuji Sato, Takashi Kikukawa |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
alpha-Defensins
Gene Expression Biology medicine.disease_cause Protein Refolding Inclusion bodies law.invention Mice Cryptdin law Escherichia coli medicine Coexpression Animals Refolding Cloning Molecular Nuclear Magnetic Resonance Biomolecular Inclusion Bodies Functional analysis Chromatography Ion Exchange NMR Recombinant Proteins Protein tertiary structure Anti-Bacterial Agents Protein Structure Tertiary Solubility Biochemistry Solubilization Yield (chemistry) Recombinant DNA α-Defensin Biotechnology |
| Zdroj: | Protein Expression and Purification. 112:21-28 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2015.04.007 |
| Popis: | Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coil expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect