[논문]Isolation of Cysteine Protease Actinidin Gene from Chinese Wild Kiwifruit and its Expression in Escherichia coli

Lee, Nam-Keun; Hahm, Young-Tae

[국내논문] Isolation of Cysteine Protease Actinidin Gene from Chinese Wild Kiwifruit and its Expression in Escherichia coli 원문보기

Food science and biotechnology, v.16 no.2 = no.74, 2007년, pp.294 - 298

Lee, Nam-Keun (Department of Biotechnology, Chung Ang University) , Hahm, Young-Tae (Department of Biotechnology, Chung Ang University)

Abstract ▼ AI-Helper

The actinidin (EC 3.4.22. 14) found in kiwifruit is a cysteine protease. In order to obtain the actinidin gene from the Chinese wild kiwifruit, primers were designed on the basis of the actinidin gene of Actinidia deliciosa, the New Zealand kiwifruit. The 1.2 kb DNA fragment was acquired from the total RNAs of Chinese wild kiwifruit via reverse transcription polymerase chain reaction (RT-PCR), and its DNA sequence was analyzed. Its sequence was determined to share 98.4% homology with the actinidin gene of A. deliciosa. In order to verity the actinidin gene isolated from the Chinese wild kiwifruit in Escherichia coli, the mature gene was amplified via PCR and expressed in E. coli under the control of the T7lac promoter. The actinidin was expressed in E. coli as inclusion bodies, which were solubilized with urea and refolded. The protease activity of the refolded protein was approximately twice as high as that of E. coli BL2l (DE3).

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제안 방법

As kiwifruit was reported to harbor a strong protease (1), research has been conducted into its molecular biological properties (2-5) and its p_니Hfication from pulp (6-9).

이론/모형

Using bovine serum albumin (BSA; Fisher Scientific, Pittsburgh, PA, USA) as a standard, the protein concentrations were determined via the Bio-Rad Bradford method.
The solubilization of inclusion bodies was conducted using a modified version of Margetts's method (22). The expressed protein was denatured with 8 M urea containing 0.
Specific protease activity was determined via a modified version of Yamaguchi's method (16, 17). One tenth %(w/v) casein in 0.

참고문헌 (23)

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원문보기 상세보기
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