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Molecules 2014, 19(10), 16179-16189; doi:10.3390/molecules191016179

Recombinant Expressed Vector pET32a (+) S Constructed by Ligation Independent Cloning

1,†
,
1,†
,
1
,
2,* and 1,3,*
1
Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for the Nationalities, Tongliao 028000, Inner Mongolia, China
2
State Key Laboratory of Medical Microbiology and Biosafety, Academy of Military Medical Sciences, Biotechnology Institute of Beijing, Beijing 100071, China
3
Institute of Mongolia and Western Medicinal Treatment, Affiliated Hospital of Inner Mongolia University for the Nationalities, Holin he Street 1742, Tongliao 028000, Inner Mongolia, China
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 5 August 2014 / Revised: 9 September 2014 / Accepted: 9 September 2014 / Published: 10 October 2014
(This article belongs to the Section Molecular Diversity)
View Full-Text   |   Download PDF [1911 KB, uploaded 10 October 2014]   |  

Abstract

The aim of this work was to develop a new method for constructing vectors, named ligation-independent cloning (LIC) method. We constructed the S label expression vector and recombinant pET32a (+) S-phoN2 by LIC. The recombinant proteins were expressed in E. coli at a high level, and then the specificity of the recombinant proteins was identified by western blot. The target band was detected by S monoclonal antibody and Apyrase polyclonal antibodies but not Trx monoclonal antibody and HIS monoclonal antibody. Finally, we obtained protein Apyrase in E. coli (BL21), with a protein-only expression S tag. Collectively, our results demonstrated that LIC is effective for the construction of new vectors and recombinant plasmids. Free from the limitations of restriction enzyme sites and with a higher positive rate, LIC processes should find broad applications in molecular biology research. View Full-Text
Keywords: ligation-independent cloning; vector constructing; prokaryotic gene expression vector ligation-independent cloning; vector constructing; prokaryotic gene expression vector
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Wang, Y.; Gong, G.-H.; Wei, C.-X.; Liang, L.; Zhang, B. Recombinant Expressed Vector pET32a (+) S Constructed by Ligation Independent Cloning. Molecules 2014, 19, 16179-16189.

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