title: Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.
abstract: We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR"). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag.
authors.affiliation: Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India.
authors.forename: Manika Indrajit
authors.initials: MI
authors.lastname: Singh
authors.forename: Vikas
authors.initials: V
authors.lastname: Jain
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D049452:Green Fluorescent Proteins / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D017931:DNA Primers; D011506:Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine