Cultivation at 6-10 ℃ is Effective to Overcome the Insolubility of Recombinant Proteins.
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Title
- Cultivation at 6-10 ℃ is Effective to Overcome the Insolubility of Recombinant Proteins.
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Author(s)
- 안영준; 김민규; 이상민; 정창숙; 차선신
- KIOST Author(s)
- An, Young Jun(안영준)
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Alternative Author(s)
- 안영준; 김민규; 이상민; 정창숙; 차선신
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Publication Year
- 2012-09-21
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Abstract
- Protein expression in Escherichia coli at 15–25 ℃ is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6–10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2 (DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 L culture grown at 6 and 10 ℃, respectively. Cultivation at 10 ℃ also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21 (DE3). Consequently, E. coli cultivation at 6–10 ℃ is an effective strategy for overcoming a major hurdle of the inclusion body formation. Mannanase (CaMan) and cellulase (CaCel) from Cryptopygus antarticus was purified and crystallized at 295 K. A 2.6 Å resolution data set of mannanase crystal has been collected using synchrotron radiation. The crystals belong to space group P212121 with unit-cell parameters a = 74.02, b = 82.45, c = 164.32 Å. A 2.6 Å resolution data set of cellulase crystal has been collected using synchrotron radiation. The crystals belong to space group P3121 with unit-cell parameters a = 81.71, b = 81.71, c = 89.35 Å, a= b = 90, γcombinant proteins can be expressed as soluble forms by simply lowering temperature to 6–10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2 (DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 L culture grown at 6 and 10 ℃, respectively. Cultivation at 10 ℃ also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21 (DE3). Consequently, E. coli cultivation at 6–10 ℃ is an effective strateg
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URI
- https://sciwatch.kiost.ac.kr/handle/2020.kiost/27568
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Bibliographic Citation
- 2012 Structure Festival, pp.29, 2012
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Publisher
- Korean
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Type
- Conference
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Language
- English
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