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Advancements in molecular biology could not have occurred without the introduction of Histidine and Green Fluorescent Protein (GFP) tagging. These methods aid in protein purification and the understanding of molecular and cellular processes via protein fluorescence tracking respectively. The use of these techniques are extremely common in today’s molecular biology field.
Histidine tagging is a purification method where histidine residues are added to the carboxyl or amino terminus of a target protein. When designing the recombinant protein, the histidine codons must be added in-frame to the protein of interest so that the tag is correctly translated. Six histidine residues are generally used as the tag, but anywhere between two to ten residues have been utilized. Recombinant proteins with such tags can be purified via immobilized metal affinity chromatography based on the interaction between histidine’s imidazole ring and a transition metal ion adhered to the column. Histidine has electron donor groups on its imidazole ring, which will selectively bind and form coordination bonds with the transition metals Co2+, Ni2+, Cu2+, or Zn2+. The most commonly used metal ion is Ni2+; however, Co2+ has been shown to obtain the highest purity of protein. Proteins without the histidine tag will not bind to the column and will be eluted out. Tagged proteins are retained in the column matrices until its release by adding increasing concentrations of free imidazole, which acts as a competitor molecule, or by lowering the column pH to protonate the metal ions breaking the coordination bonds. Histidine tagging provides an extremely quick and efficient method for isolating the protein of interest.
GFP was first isolated from the jellyfish Aequorea Victoria and is now used to monitor gene expression and protein localization in living organisms. Structurally, GFP forms a chromophore with its serine-dehydrotyrosine-glycine motif found in a hexapeptide starting at amino acid 64, and this sequence will optimally absorb UV light of 395 nm or blue light of 475 nm and emit bright green light of 509 nm. When GFP was cloned and expressed in Escherichia coli, the observed fluorescence means the gene and resulting protein contained all the necessary information to synthesize the chromophore and no additional enzymes or co-factors are required. GFP is fused in frame with the gene encoding an endogenous protein at the C-terminus or the N-terminus. In addition, a linker sequence can be added to promote proper folding and function of GFP and the protein of interest. A protein tagged with GFP can be visualized and tracked in living cells when under long wavelength UV light.
In a research setting, GFP tagging was used to investigate the role of SOX2 in squamous cell carcinoma (SCC) with regard to tumor initiation and cancer-stem cell functions. Mice with GFP tagged to SOX2 was obtained from Jackson Laboratories. The green florescence was used as a reporter for Sox2 transcriptional expression. By using GFP’s fluorescence, it was observed that Sox2 is an upregulated transcription factor expressed in cancer stem cells and SOX2 is normally absent in normal epidermis. Sox2 expression is enriched in tumor-propagating and tumor-initiating cells in invasive SCC. In addition, a gene network regulated by SOX2 was uncovered in primary tumor cells in vivo by using gene expression profiling. By uncovering this information and identifying the functions and downstream target gene network of SOX2, this study is relevant to ongoing cancer research in developing a strategy for treatment not only for SCC, but to the multitude of genes affected by SOX2.
Histidine and GFP tagging are important tools in advancing research in the field of molecular biology. Histidine tags are able to purify proteins extremely efficiently, and GFP tags aid in observing and tracking proteins in living cells. Ongoing research using these techniques can give a better understanding of cellular processes in organisms, which can ultimately be applied in improving health.
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