Histidine and Green Fluorescent Protein(GFP), tagging, have made molecular biology more accessible. These techniques are used to purify proteins and aid in understanding molecular processes and cellular processes using protein fluorescence monitoring. These techniques are very common in the field of molecular biology.
Histidine tagging can be used to purify proteins by adding histidines to their carboxyl or amino terminus. In order to correctly translate the histidine codeons into the target protein’s frame, it is essential that they are added when designing the recombinant. The tag is usually composed of six histidine residues. However, it has been used with up to ten residues. You can purify recombinant protein with such tags using immobilized metallic affinity chromatography. This technique uses the interaction between the column’s transition metal ion and histidine’s imidazole-ring. Histidine is a metal with electron donor groups. These will selectively bind or form coordination bonds to the transition metals Cu2+/Ni2+, Zn2+, and Ni2+. Ni2+ is the most common metal ion. However, Co2+ has been proven to be the best for protein purity. Hetidine tags are not required for proteins to bind to the column. They will be removed. To release tagged proteins, you can increase their concentrations of free imidazole or decrease the pH to protonate the metal ions. Histidine tag is an efficient and fast method to locate the desired protein. 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. GFP was cloned into Escherichia coli and expressed. The observed fluorescence meant that the gene had all the information necessary to synthesize chromophore. GFP can be fused in frame to the gene that encodes an endogenous prot at the C/N-terminus. To promote the proper folding and function, a linker sequence may be added. GFP can also be used to visualize and track a protein in living cells, when it is exposed to long-wave UV light.
GFP tag was used to explore the role SOX2 played in Squamous Cell Carcinoma (SCC) tumor initiation, and cancer-stem cells functions. Jackson Laboratories provided mice with GFP-tagged SOX2 tags. The reporter for Sox2 expression was the green fluorescence. GFP fluorescence showed that Sox2 transcription was upregulated in cancer-stem cells. Normal epidermis lacks SOX2. In invasive SCC, Sox2 expression is higher in tumor-initiating and tumor-propagating cells. Gene expression profiling was used to uncover a gene network that SOX2 regulates in primary tumor cells. This research is important for continuing cancer research. It identifies the functions of SOX2 as well as the downstream target gene networks.
GFP and histidine tags play an important role in the advancement of research in molecular biological research. GFP tags, which can track and monitor proteins in living tissues, are capable of removing proteins from cells with high efficiency using histidine tags. These techniques can be used to improve health by providing ongoing research.
