Expression Vectors for Enzyme Restriction- and Ligation-Independent Cloning for Producing Recombinant His-Fusion Proteins

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• 作者：Blanca de las Rivas ; Jos&eacute ; Antonio Curiel ; Jos&eacute ; Miguel Mancheñ ; o ; Rosario Muñ ; oz
• 刊名：Biotechnology Progress
• 出版年：2007
• 出版时间：June 2007
• 年：2007
• 卷：23
• 期：3
• 页码：680 - 686
• 全文大小：159K
• 年卷期：v.23,no.3(June 2007)
• ISSN：1520-6033

文摘

In this work we have constructed two novel expression vectors, designated as pURI2 and pURI3,which enable parallel cloning of a given target gene for producing recombinant His-fusionproteins. The vectors were created using the well-known pT7-7 and pIN-III-A3 plasmids astheir template. The same DNA fragment containing the His-tag, enterokinase cleavage site, anda NotI unique site, as well as keeping the HindIII unique restriction site, was introduced in bothvectors. These vectors have been designed to avoid the enzyme restriction and ligation stepsduring the cloning. The unique NotI site was introduced to facilitate the selection of the adequaterecombinant plasmid. Parallel cloning of the same polymerase chain reaction fragment can becarried out since both vectors shared the same leader sequence. The described strategy avoidstedious cloning efforts into different expression vectors and represents a highly efficient meansof cloning. To validate our vectors, we have cloned one target gene in both vectors and usedexpression and purification techniques to obtain the recombinant target protein. We herein showthat both vectors function effectively in all the required experimental steps-cloning, expression,purification, and cleavage.