Expressing and Purifying the His-Tagged Lipase Protein from Candida Rugosa

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Project title

Code/計畫編號

NSC86-2313-B019-002

Translated Name/計畫中文名

Candida Rugosa標籤後的脂肪酵素的表現與純化

Project Coordinator/計畫主持人

Shye-Jye Tang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=306783

Year

1997

Start date/計畫起

01-08-1996

Expected Completion/計畫迄

01-07-1997

Bugetid/研究經費

503千元

ResearchField/研究領域

生物技術(理)

Candida rugosa產生的脂肪酵素(CRL),廣泛用於食品、清潔劑和藥物及精密化學品的合成上,該酵素並具有膽固醇水解酵素活性,廣用於臨床檢驗上。CRL基因已有七種被發表,唯該CTG codon在C. rugosa產生Ser而非Leu,因此無法在常使用的蛋白質表現系統表現出活性。本實驗計畫,使用Zeocin為C. rugosa的Dominant selection marker。為使Zeocin能在C. rugosa表現出活性,使用PCR技術,將其上CTG codon修改為Leu codon。這基因重組zeocin,經送入E. Coli and yeast,可測出活性和蛋白質表現。這顯示基因重組Zeocin仍具有功能。未來將建立C. rugosa表現載體,接入CRL基因,以電破法轉殖入C. rugosa。這可建立特殊C. rugosa表現系統。因此,將有利於單一純C. rugosa脂肪酵素的製備、純化和新用途的開發和研究。 Candida rugosa produces extracellular lipases (CRL) that have been extensively applicated in the food or detergent industries, and the synthesis of the pharmaceuticals and fine chemicals etc.. The CRL supplied by different manufacturers under various culture conditions showed five distinct activity bands on gel, suggesting the existence of multiple forms. Moreover, eight coding sequences of lipases have been identified from Candida rugosa. By comparing with protein sequence, molecular weight or physical properties, those lipases show highly similarity. Therefore, it is difficult to purify single product from a crude mixture. In contrast codon usage, CTG is encodes a Ser in Candida rugosa, but universal codon is encoded a Leu. Attempt to produce these polypeptides in enzymatically active form in E. coli or yeast revealed different codon usage toward the host. Thus, to obtain the recombinant lipase from E. coli or yeast is difficult. Overwhelming the codon usage and simplifying the expression of recombinant lipase are able to express lipase in the Candida rugosa using dominant selection maker, Zeo, which codon are corrected by anneal-extension PCR method in order to fit C. rugosa codon usage. Recombinant Zeo gene (rZeo) ligated into E. coli expression vector, pET expression system, was transformed into AD494 (DE3), and was selected by Zeocin agar plate. Then, the transformants, resist to zeocin, appear the zeo protein analysing by SDS-PAGE, indicating that the rZeo have correct open-reading frame and reveal activity. Subsequently, the rZeo was cloned into yeast expression vector and transform into C. rugosa using Lithium acetate method. Zeocin-resistant clones were collected and shown the rZeo gene integrated into chromosome of C. rugosa by PCR. Our experiments show that the model of transformation and selection was established that will apply to express recombinant product into C. rugosa.

Keyword(s)

脂肪酶
蛋白質表現
選殖
純化
Lipase
Protein expression
Cloning
Purification
Candida rugosa

DSpace CRIS

Expressing and Purifying the His-Tagged Lipase Protein from Candida Rugosa

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