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Expression Vectors for Enzyme Restriction‐ and Ligation‐Independent Cloning for Producing Recombinant His‐Fusion Proteins
Author(s) -
Rivas Blanca de las,
Curiel José Antonio,
Miguel Mancheño José,
Muñoz Rosario
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp060318g
Subject(s) - restriction enzyme , cloning (programming) , recombinant dna , multiple cloning site , restriction site , fusion protein , plasmid , cloning vector , biology , molecular cloning , expression vector , in vitro recombination , microbiology and biotechnology , restriction digest , ligation , computational biology , genetics , vector (molecular biology) , gene , gene expression , computer science , programming language
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‐fusion proteins. The vectors were created using the well‐known pT7–7 and pIN‐III‐A3 plasmids as their template. The same DNA fragment containing the His‐tag, enterokinase cleavage site, and a Not I unique site, as well as keeping the Hin dIII unique restriction site, was introduced in both vectors. These vectors have been designed to avoid the enzyme restriction and ligation steps during the cloning. The unique Not I site was introduced to facilitate the selection of the adequate recombinant plasmid. Parallel cloning of the same polymerase chain reaction fragment can be carried out since both vectors shared the same leader sequence. The described strategy avoids tedious cloning efforts into different expression vectors and represents a highly efficient means of cloning. To validate our vectors, we have cloned one target gene in both vectors and used expression and purification techniques to obtain the recombinant target protein. We herein show that both vectors function effectively in all the required experimental steps‐cloning, expression, purification, and cleavage.