Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography
| Autor: | Xin Tong, Hai Hong Wang, Sheng Bin Wang, Qi Hang Yuan, Can Bin Ouyang, Hua Zhen Wang, Zhong Kun Wu, Chang Chao Chen, Mi Nan Wang, Ao Cheng Cao, Zhi Zhan Chu |
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| Rok vydání: | 2015 |
| Předmět: |
Proteases
medicine.medical_treatment Recombinant Fusion Proteins lcsh:Medicine Chromatography Affinity Affinity chromatography Protein purification Endopeptidases Glucokinase medicine Escherichia coli lcsh:Science Tandem affinity purification Multidisciplinary Protease biology Escherichia coli Proteins lcsh:R Fusion protein Recombinant Proteins Acyl carrier protein Biochemistry Solubility biology.protein lcsh:Q Target protein alpha-Amylases Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 10, Iss 12, p e0143598 (2015) |
| ISSN: | 1932-6203 |
| Popis: | Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. |
| Databáze: | OpenAIRE |
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