PRIME (PRobe Incorporation Mediated by Enzymes)

PRIME (PRobe Incorporation Mediated by Enzymes) is a molecular biology research tool developed by Alice Y. Ting and the Ting Lab at MIT for site-specific labeling of proteins in living cells with chemical probes.[1][2] Probes often have useful biophysical properties, such as fluorescence, and allow imaging of proteins.[1] 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.[3] 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 (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.[3] 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.[4] PRIME has been developed in order to achieve a high labeling specificity comparable to fluorescent proteins with small molecules.[4]

Principles

In PRIME, a mutant enzyme LplA (lipoic acid ligase from Escherichia coli) first catalyzes the conjugation of the “functional group handle” and LplA acceptor peptide (LAP), which is genetically fused to the protein of interest.[1][4][5] “Functional group handle” indicates a bridge molecule connecting a LAP tag to a fluorescent probe or fluorophore. 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,[6] and chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC) (refer to Azide-alkyne Huisgen cycloaddition).[7] Two other versions of PRIME labeling technologies use mutant LplA proteins to directly incorporate a fluorophore to the LAP-tagged protein of interest.[8][9]

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.[1] It is recommended that the experimenters perform control experiments in order to make sure that the tagged recombinant protein functions properly.[1] Secondly, even at a low concentration, chemicals such as the fluorescent probe can be toxic to the cells.[1] Experimenters are also required to obtain the right balance between maximal signal of fluorescence and minimal disruption of cellular function.[1]

References

  1. 1 2 3 4 5 6 7 Uttamapinant C, Sanchez MI, Liu DS, Yao JZ, Ting AY (August 2013). "Site-specific protein labeling using PRIME and chelation-assisted click chemistry". Nat Protoc. 8 (8): 1620–34. doi:10.1038/nprot.2013.096. PMID 23887180.
  2. ↑ Tschesche H, ed. (2011). Methods in protein biochemistry. Berlin: De Gruyter. ISBN 978-3-11-025236-1.
  3. 1 2 Soh N (19 February 2008). "Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology". Sensors. 8 (2): 1004–1024. doi:10.3390/s8021004.
  4. 1 2 3 Yao JZ, Uttamapinant C, Poloukhtine A, Baskin JM, Codelli JA, Sletten EM, Bertozzi CR, Popik VV, Ting AY (2012). "Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition". J. Am. Chem. Soc. 134 (8): 3720–8. doi:10.1021/ja208090p. PMC 3306817Freely accessible. PMID 22239252.
  5. ↑ Demchenko AP, Brouwer AM, ed. (2011). Advanced fluorescence reporters in chemistry and biology. Heidelberg: Springer. ISBN 978-3-642-18034-7.
  6. ↑ Liu DS, Tangpeerachaikul A, Selvaraj R, Taylor MT, Fox JM, Ting AY (2012). "Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells". J. Am. Chem. Soc. 134 (2): 792–5. doi:10.1021/ja209325n. PMC 3381951Freely accessible. PMID 22176354.
  7. ↑ Uttamapinant C, Tangpeerachaikul A, Grecian S, Clarke S, Singh U, Slade P, Gee KR, Ting AY (2012). "Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling". Angew. Chem. Int. Ed. Engl. 51 (24): 5852–6. doi:10.1002/anie.201108181. PMC 3517120Freely accessible. PMID 22555882.
  8. ↑ Uttamapinant C, White KA, Baruah H, Thompson S, Fernandez-Suarez M, Puthenveetil S, Ting AY (2010). "A fluorophore ligase for site-specific protein labeling inside living cells.". Proc Natl Acad Sci U S A. 107 (24): 10914–9. doi:10.1073/pnas.0914067107. PMC 2890758Freely accessible. PMID 20534555.
  9. ↑ Liu DS, Nivon LG, Richter F, Goldman PJ, Deerinck TJ, Yao JZ, Phipps WS, Ellisman MH, Drennan CL, Baker D, Ting AY (2014). "Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.". Proc Natl Acad Sci U S A. 111 (43): E4551–9. doi:10.1073/pnas.1404736111. PMC 4217414Freely accessible. PMID 25313043.
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