ArticlePloS one2013
Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 21 citations in OpenAlex.
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- Deciphering the molecular mechanism and regulation of formaldehyde detoxification inApplied and environmental microbiology · 2024Article
- A positive SelectionFrontiers in bioengineering and biotechnology · 2021Article
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- MnoSR Is a Bona Fide Two-Component System Involved in Methylotrophic Metabolism in Mycobacterium smegmatis.Applied and environmental microbiology · 2019Article
- Methylotrophy in Mycobacteria: Dissection of the Methanol Metabolism Pathway in Mycobacterium smegmatis.Journal of bacteriology · 2018Article
- Identification and Characterization of an Inside-Out Folding Intermediate of T4 Phage Sliding Clamp.Biophysical journal · 2017Article
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- Molecular dissection of phage endolysin: an interdomain interaction confers host specificity in Lysin A of Mycobacterium phage D29.The Journal of biological chemistry · 2014Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.