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1.
Methods Mol Biol ; 1266: 145-59, 2015.
Article in English | MEDLINE | ID: mdl-25560073

ABSTRACT

Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment. In the resulting spontaneous protein trans-splicing reaction the split intein fragments remove themselves and ligate the tag to the protein of interest in a virtually traceless fashion. The reaction can be performed either using a purified protein of interest or to label a protein in the context of a living cell. All protein components are recombinantly expressed and all chemical reagents are commercially available.


Subject(s)
Inteins , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Animals , Cell Line , Click Chemistry , Escherichia coli , Mice , Protein Engineering , Protein Splicing , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Staining and Labeling
2.
Angew Chem Int Ed Engl ; 53(16): 4113-7, 2014 Apr 14.
Article in English | MEDLINE | ID: mdl-24615830

ABSTRACT

Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans-splicing. For the amine-tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine-reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click-tag, protein trans-splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p-azidophenylalanine. By the two-step strategy, exposure of the protein of interest to the copper catalyst was avoided.


Subject(s)
Amines/chemistry , In Vitro Techniques/methods , Protein Splicing/genetics , Proteins/chemistry , Humans , Inteins/genetics
3.
FEBS Lett ; 583(5): 909-14, 2009 Mar 04.
Article in English | MEDLINE | ID: mdl-19302791

ABSTRACT

We have studied the naturally split alpha subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and determined an apparent first-order rate constant of (1.1+/-0.2)x10(-2) s(-1) at 37 degrees C. This represents the highest rate reported for the protein trans-splicing reaction so far (t(1/2) of approximately 60s). Furthermore, the reaction was very robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37 degrees C, and the presence of up to 6 M urea. Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chemistry.


Subject(s)
DNA Polymerase III/metabolism , Inteins , Nostoc/genetics , Nostoc/metabolism , Trans-Splicing/genetics , DNA Polymerase III/genetics , Gene Expression , Point Mutation/genetics , Time Factors
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