Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.
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 f...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4671538?pdf=render |
| _version_ | 1818129284911333376 |
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| author | Hua-zhen Wang Zhi-zhan Chu Chang-chao Chen Ao-cheng Cao Xin Tong Can-bin Ouyang Qi-hang Yuan Mi-nan Wang Zhong-kun Wu Hai-hong Wang Sheng-bin Wang |
| author_facet | Hua-zhen Wang Zhi-zhan Chu Chang-chao Chen Ao-cheng Cao Xin Tong Can-bin Ouyang Qi-hang Yuan Mi-nan Wang Zhong-kun Wu Hai-hong Wang Sheng-bin Wang |
| author_sort | Hua-zhen Wang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-11T07:46:42Z |
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| id | doaj.art-0f53cf2d1e9948d7b977753907a706a7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T07:46:42Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-0f53cf2d1e9948d7b977753907a706a72022-12-22T01:15:26ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-011012e014359810.1371/journal.pone.0143598Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.Hua-zhen WangZhi-zhan ChuChang-chao ChenAo-cheng CaoXin TongCan-bin OuyangQi-hang YuanMi-nan WangZhong-kun WuHai-hong WangSheng-bin WangFusion 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.http://europepmc.org/articles/PMC4671538?pdf=render |
| spellingShingle | Hua-zhen Wang Zhi-zhan Chu Chang-chao Chen Ao-cheng Cao Xin Tong Can-bin Ouyang Qi-hang Yuan Mi-nan Wang Zhong-kun Wu Hai-hong Wang Sheng-bin Wang Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography. PLoS ONE |
| title | Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography. |
| title_full | Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography. |
| title_fullStr | Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography. |
| title_full_unstemmed | Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography. |
| title_short | Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography. |
| title_sort | recombinant passenger proteins can be conveniently purified by one step affinity chromatography |
| url | http://europepmc.org/articles/PMC4671538?pdf=render |
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