Anton Simeonov Ph.D. National Institute of Health
Protein Tagging Is Widely Used In Approaches Ranging From Affinity Purification to Fluorescence-Based Detection in Live Cells
Expressed protein fusion tags comprise amino acid sequences, ranging from short peptides, such as FLAG and hexahistidine tails, to full-size proteins, such as glutathione S-transferase, appended to the N- or C-terminus of a protein of interest through a genetic construct. By virtue of having the defined tag as an integral part of the protein of interest, one can easily track or sequester/purify the protein. The discovery and adoption of fusion tags has not always followed a rational strategy, with both very short and relatively long sequences dominating the range of available tags. While these existing tags have enjoyed an extremely wide adoption, they carry specific drawbacks: the very short tags represent peptides that can adopt disordered structures and thus trigger misfolding of the protein under study, while the full-length protein tags add a burden to the cellular machinery when the sometimes too-large fusion protein needs to be produced. The present work provides an elegant solution to this problem in the form of relatively short peptide sequences of an approximate size of a few dozen amino acids that have been selected for their ability to adopt well-defined tertiary structures (see figure). The authors term these “inntags” because of their innocuous nature. Inntag design was initiated by a search for (small) protein domains of specific three-dimensional structure, with further filters applied computationally, followed by specific point modifications to arrive at several candidates. The utility of the new inntags was evaluated through various measures of proper fusion protein folding, intracellular localization, and overall cell fitness. Since the sequences of the inntags have been made publicly available, a further validation and rapid adoption is expected.
Cell fitness effects and interactome analysis of selected inntags and other commonly used tags. (a) Epitope tags may have diverse, deleterious structural and functional effects on target proteins. Tags can interfere with proper folding of target proteins and cause aggregation or degradation of misfolded intermediates or drive spurious conformational changes that alter the function or intracellular localization of the target protein. (b) Structures of inntags IT5 (PDB 1WHP), IT6 (PDB 1WKX) and IT10 (PDB 3BBG) selected by bioinformatics screening. (c) Disorder predisposition of tags at the amino acid resolution level. (d) GFP fusions to different tags were overexpressed in budding yeast cells to determine individual critical volumes at budding (N>400). Mean values (thick vertical lines) and confidence limits (α=0.05, thin vertical lines) for the mean are also shown. (e) iTRAQ analysis of proteins interacting with MYC6 and IT6 GO terms and the corresponding uncorrected P values (–log) of yeast proteins interacting with MYC6 or IT6 fused to His6-GFP and identified by iTRAQ. (f) iTRAQ ratios obtained for MYC6 and IT6 interactors belonging to the family of molecular chaperones (Hsp60, Sse1, Ssa1, Ssa2, Ssb2, Ssc1, Sti1, Aha1, Ydj1 and Sis1). Mean enrichment values (thick horizontal lines) relative to untagged GFP and confidence limits (α=0.05, thin horizontal lines) for the mean are also shown. (g) Yeast cells overexpressing GFP fused to MYC6 and IT6 were analyzed by time-lapse confocal microscopy under FLIP conditions. Protein mobility mean values (N=40 cells) and confidence limits for the mean (α=0.05) are shown. r.u., relative units. Comparable quantitative differences were obtained from a replicate experiment (d,g); statistical data were obtained from three independent pulldown experiments (e,f). For a larger image click here.
* Abstract from Nat Methods 2015;12:955–958
Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. However, an intrinsic limitation of tagging is that the native function of the protein may be compromised or even abolished by the presence of the tag. Here we describe and characterize a set of small, innocuous protein tags (inntags) that we anticipate will find application in a variety of biological techniques.
Anton Simeonov, Ph.D., works at the NIH
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Nature Methods titled "Inntags: small self-structured epitopes for innocuous protein tagging." Authors of the paper are Maya V Georgieva, Yahya G, Codó L, Ortiz R, Teixidó L, Claros J, Jara R, Jara M, Iborra A, Gelpí JL, Gallego C, Orozco M, Aldea M.
