Protein analysis techniques: Histidine and Green Fluorescence Protein tagging
The shift into the post-genomic era also saw a shift from the analysis of genomic sequences, to the analysis of the proteins they encode (Waugh, 2005). Histidine-tagging and Green Fluorescence Protein (GFP) tagging are two technologies developed to aid in the discovery of protein function.
Histidine-tagging involves the usage of immobilized metal-affinity chromatography (IMAC) and the attachment of typically six polyhistidine residues to the termini of recombinant proteins (Mathias et al., 2010). The initial attachment of the histidine (His) residues to the target protein is convenient, as the small size allows easy integration into all expression vectors via site-directed mutagenesis, or by polymerase chain reaction (PCR) methods (Mathias et al., 2010). The purification process involves the attachment of the tagged protein to an absorbent consisting of immobilized ions (i.e., IMAC) as the His residues have a high affinity to divalent metal ions (Shao et al., 2018). The untagged proteins are then washed with other solvents, while the tagged protein is eluted with imidazole (Shao et al., 2018).
The utilization of this process has been a catalyst in protein purification (Waugh, 2005). The current post-genomic era brought a surge of proteins produced from recombinant techniques, and with it, the need for rapid, high throughput screening approaches (Arnau et al., 2006). These screening approaches are used to identify potential therapeutic, diagnostic, or industrial proteins for application in medical and scientific fields (Arnau et al., 2006).
There are many areas of the His tag process that can be customized to fit the specific needs of each target protein. In regards to the length of the polyhistidine residues, although His tags of less or more than six had been successfully utilized, six residues have been found to be the optimal number for efficient purification; as additional residues may affect protein function, while less residues may affect proper purification (Mathias et al., 2010). The position of attachment (i.e., C- or N- terminal) is dependent upon the protein, and can be changed to enhance the tagging ability to the column (Mathias et al., 2010). The specific binding of His to divalent metal ions, particularly Cu, Ni, Co, and Zn, is a characteristic utilized not only in protein purification but also in the attachment of proteins or peptides to micro- and nanoparticles (Tsai et al., 2014). The purification process has additionally been improved by media products coupled to a solid support resin, e.g., nickel-nitrilotriacetic acid (Ni2+-NTA) and Co2+-carboxylmethylaspartate (Co2+-CMA,) which aid with metal to histidine interactions (Mathias et al., 2010).
GFP, a protein isolated from the jellyfish, Aequorea victoria, is naturally fluorescent and is another analysis method utilized in protein labelling to follow the movement of fusion proteins (Yuste, 2005). The gene can be incorporated and expressed in any species that can be genetically engineered (Yuste, 2005).
GFP tagging is utilized in many applications. Its fluorescent property is used for fluorescence microscopy, a technique helpful in the visualization of all biological specimen, as well as for the tracking of the labeled proteins (Yuste, 2005). This aided in the discovery of gene expression, protein localization, protein–protein interactions, calcium concentrations, and redox potentials (Deponte, 2012). Another useful feature of GFP is its ability to form intrinsic chromophores in living cells without the need for cofactors, optimizing the transfection of the gene, and with it, the ability for a myriad of cells to fluoresce (Stepanenko et al., 2008).
Like His tagging, the optimization of certain areas of GFP tagging can enhance the analysis technique. The location of the tag, in terms of which terminal end of the protein it is found in, is imperative to the preservation of target protein localization, as C-terminal tagging was found to be better in comparison to N-terminal tagging (Palmer and Freeman, 2004). The size of GFP matters as well (Snapp, 2005). The introduction of the tag is not necessarily a small one due its 27 kDa size, and therefore protein behavior must be analyzed for any formation of aggregates, and the size or site of attachment must be changed accordingly (Snapp, 2005). Additionally, site-directed mutagenesis of GFP has allowed for the shifts of its typical UV spectrum of ~509nm, thus allowing shifts in colour as well (Gurskaya et al., 2001; Snapp, 2005). E.g., a substitution of GFP’s Tyr-66 with any aromatic residue resulted in a blue colour shift (λmax = 442 nm) (Gurskaya et al., 2001).
An example of research that utilized one of these techniques was conducted for the visualization of function and cross-species activity in Drosophila (Tsuda et al., 2015). The function of sex-peptide (SP) orthologs are not very well known in the Drosophila species, in regards to whether these functions are conserved or specific to each species (Tsuda et al., 2015). In order to visualize the binding of SP to its receptors across various species, Drosophila melanogaster sex-peptide (melSP), a seminal fluid that induces postmating responses (PMR) via sex-peptide receptor (SPR) was GFP-tagged (Tsuda et al., 2015). It was found that only D. melanogaster employs SP-mediated PMR with high expression levels of SPR in the oviducts (Tsuda et al., 2015). This higher expression levels suggests that it plays an important part in the establishment of SP-mediated PMR throughout the species’ evolution (Tsuda et al., 2015).
Therefore, the employment of His and GFP tagging in protein analysis techniques have been a large benefit across many fields. The customization of these techniques, e.g., site-directed mutagenesis, as well as the development of new methods and products can only widen its usefulness in the discovery of various protein function.
Arnau J, Lauritzen C, Petersen G E, Pedersen J (2006). Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48, 1–13.
Deponte M (2012). GFP tagging sheds light on protein translocation: Implications for key methods in cell biology. Cell Mol Life Sci 69, 1025–1033.
Gurskaya N G, Savitsky A P, Yanushevich Y G, Lukyanov S A, Lukyanov K A (2001). Color transitions in coral’s fluorescent proteins by site-directed mutagenesis. BMC Biochem 2, 1–7.
Mathias J R, Dodd M E, Walters K B, Yoo S K, Erik A, Huttenlocher A (2010). Purification of Proteins Using Polyhistidine Affinity Tags. 33, 1212–1217.
Palmer E, Freeman T (2004). Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays. Comp Funct Genomics 5, 342–353.
Shao M, Xiu L, Zhang H, Huang J, Gong X (2018). Chitosan/cellulose-based beads for the affinity purification of histidine-tagged proteins. Prep Biochem Biotechnol 48, 352–360.
Snapp E (2005). Design and Use of Fluorescent Fusion Proteins in Cell Biology. Curr Protoc Cell Biol 27, 1–17.
Stepanenko O, Verkhusha V, Kuznetsova I, Uversky V, Turoverov K (2008). Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes. Curr Protein Pept Sci 9, 338–369.
Tsai H Y, Lee A, Peng W, Yates M Z (2014). Synthesis of poly(N-isopropylacrylamide) particles for metal affinity binding of peptides. Colloids Surfaces B Biointerfaces 114, 104–110.
Tsuda M, Peyre J B, Asano T, Aigaki T (2015). Visualizing molecular functions and cross-species activity of sex-peptide in Drosophila. Genetics 200, 1161–1169.
Waugh D S (2005). Making the most of affinity tags. Trends Biotechnol 23, 316–320.
Yuste R (2005). Fluorescence microscopy today. Nat Methods 2, 902–904.
Cite This Work
To export a reference to this article please select a referencing stye below:
Related ServicesView all
Related ContentAll Tags
Content relating to: "Chemistry"
Chemistry is a science involving the study of the elements and matter at the atomic and molecular level including their composition, structure, properties, behaviour, and how they react or combine.
Processes of Drugs Metabolism in the Body
Abstract Metabolism of drugs is a complex and major process within the body, occurring primarily in the liver. The aim of metabolism is to make the drug more polar to enable excretion via the kidneys....
Monochloramine to Eliminate Biofilm in Water Treatment System
This study eluates the prospective use of monochloramine to eliminate potential biofilm in a potable water and cooling tower water treatment system....
DMCA / Removal Request
If you are the original writer of this dissertation and no longer wish to have your work published on the UKDiss.com website then please: