PRIME (probe incorporation mediated by enzymes) is a

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

research tool developed by Alice Y. Ting and the Ting Lab at

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for site-specific labeling of proteins in living cells with chemical probes. Probes often have useful biophysical properties, such as

fluorescence Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with colore ...

, and allow imaging of proteins. Ultimately, PRIME enables scientists to study functions of specific proteins of interest.

Significance

Protein labeling with fluorescent molecules allows the visualization of protein dynamics, localization, and protein-protein interactions, and therefore serves as an important technique to understand protein functions and networks in living cells. The protein labeling should have a high selectivity towards the protein of interest, and should not interfere with the natural functions of the protein. Although genetic coding of fluorescent proteins, such as the

green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea victo ...

(GFP), is the most popular technique due to its high specificity, fluorescent proteins are likely to interfere with the functions of the protein to which they are fused because of their large sizes. There are multiple tagging tools, such as HaloTag, SNAP tag, and FlAsH, developed in order to overcome the weakness of traditional protein labeling with fluorescent proteins. However, they still have significant shortcomings either due to the large size of a tag or the low specificity of the labeling process. PRIME has been developed in order to achieve a high labeling specificity comparable to fluorescent proteins with small molecules.

Principles

In PRIME, a

mutant In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It i ...

enzyme LplA (lipoic acid ligase from ''

Escherichia coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...

'') first catalyzes the conjugation of the "functional group handle" and LplA acceptor

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

(LAP), which is genetically fused to the protein of interest. “Functional group handle” indicates a bridge molecule connecting a LAP tag to a fluorescent probe or

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

. Fluorescent probe reacts with the “functional group handle” connected to the tag, and ultimately labels the protein of interest. Different chemical reactions can be utilized to attach the fluorescent probe to a complex consisting of the protein, the LAP tag, and the bridge: Diels-Alder Reaction, and chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC) (refer to

Azide-alkyne Huisgen cycloaddition The azide-alkyne Huisgen cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole. Rolf Huisgen was the first to understand the scope of this organic reaction. American chemist Ka ...

). Two other versions of PRIME labeling technologies use mutant LplA proteins to directly incorporate a fluorophore to the LAP-tagged protein of interest.

Limitations

Despite the advantages of PRIME over other tagging methods, PRIME still has some possible limitations. First of all, the LAP tag may interfere with the function of proteins to which it is fused. It is recommended that the experimenters perform control experiments in order to make sure that the tagged recombinant protein functions properly. Secondly, even at a low concentration, chemicals such as the fluorescent probe can be toxic to the cells. Experimenters are also required to obtain the right balance between maximal signal of fluorescence and minimal disruption of cellular function.

References

{{Reflist, 35em Molecular biology techniques Cell imaging Protein imaging