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'Traceless’ protein tracing

January 2016.  Protein trans-splicing mediated by split inteins is a powerful methodology for site-specific protein modification. Despite recent developments there is however still an urgent need for ultra-small high-affinity intein tags for in vitro and in vivo approaches. So far only very few in-cell applications of protein trans-splicing have been reported and all are limited to C-terminal protein modifications. A team of Frankfurt scientists led by Robert Tampé has developed a new strategy for covalent N-terminal intein-mediated protein labeling at (sub)nanomolar probe concentrations. The new strategy has just been published by the journal Chemical Science. Combined with a minimal synthetic lock-and-key element, the affinity between the intein fragments was increased to 10 nM. The team demonstrated site-specific and efficient 'traceless' protein modification by high-affinity trans-splicing at nanomolar concentrations in living mammalian cells.

Although the use of fluorescent protein has provided  fundamental  insights  into protein function and cellular processes, their  applicability is limited by their large size, slow maturation time, low photostability and quantum yield. Alternative  approaches have been developed to introduce synthetic fluorescent probes with improved photo-physical properties by self-labeling proteins but in these approaches the enzymes need to be supplied at high concentrations (1-100 μM) and the large fusion domains (≥ 20 kDa) can have adverse effects on the function, interaction and trafficking of the target protein. Besides these strategies, different chemoselective reactions and semi-synthetic techniques have been established, such as native chemical ligation or intein-mediated  protein splicing. Inteins are internal protein domains, which facilitate their own excision and thereby covalently fuse the flanking N- and C-terminal exteins in a self-processive manner.

A very promising strategy for protein semi-synthesis is protein trans-splicing (PTS), which has been applied for segmental isotope labeling, protein backbone cyclization, cyclic peptide generation, protein immobilization, as well as for N- and C-terminal protein labeling. In PTS, the autocatalytic domain is naturally or artificially split into two fragments, reconstituting the active intein complex. The excision process is virtually traceless, apart from a few flanking extein residues, required for efficient splicing. Whereas semi-synthetic PTS is widely applied in vitro,  in-cell applications using split inteins have been exclusively based on C-terminal protein modifications by trans-splicing. If the native C-terminus is not accessible or essential for function, localization or protein dynamics (e.g. lipid- and tail-anchored proteins, ubiquitin, lamin A), intracellular N-terminal protein modifications by PTS are required. However, the large sizes of most split intein fragments with 100-130 amino acids for  the N-terminal intein fragment  and 35-50 amino acids for the C-terminal intein fragment, also including high-affinity inteins compromise their accessibility by solid-phase peptide synthesis (SPPS). 

The Frankfurt scientists adopted  the  multivalent chelator tris-N-nitrilotriacetic acid (trisNTA), which allows for site-specific and reversible recognition of His6-10-tagged proteins in the nanomolar range, even inside living cells. The trisNTA/His-tag system defines one of the smallest high-affinity recognition elements known to date. The team developed a split intein  system guided by this minimalistic interaction pair to promote N-terminal protein labeling at nanomolar concentrations by trans-splicing. The new approach combines this diminutive interaction pair with the smallest synthetically accessible N-terminal intein fragment (11 amino acids) and a recombinantly expressed C-terminal intein fragment (143 amino acids) of an artificially split mini-intein from the bacterium Synechocystis sp. More ...

Robert Tampé, Institute of Biochemistry, tampe@em.uni-frankfurt.de
Goethe University Frankfurt, Max von Laue Straße 9, 60438 Frankfurt/Germany


M. Braner, A. Kollmannsperger, R. Wieneke and R. Tampé (2015) ’Traceless’ tracing of proteins – High-affinity trans-splicing directed by a minimal interaction pair. Chem. Sci., published online 21 Dec 2015, DOI: 10.1039/C5SC02936. Link