In vitro protein splicing of purified precursor and the identification of a branched intermediate

Xu, Ming‐Qun; Southworth, Maurice W.; Mersha, Fana B.; Hornstra, Linda J.; Perler, Francine B.

doi:10.1016/0092-8674(93)90623-x

Cited by 201 publications

(234 citation statements)

References 19 publications

Supporting

Mentioning

216

Contrasting

Unclassified

Order By: Relevance

“…When an intein BI accumulates, the (thio)ester linkage is usually alkaline labile at elevated temperatures. 5,[18][19][20] The affinity purified Asn343Ala BI was incubated overnight in vitro at 4 C, room temperature, 30 C or 37 C, pH 6 or 9, and in the presence or absence of 50 mM DTT [ Fig. 5(A) and data not shown].…”

Section: Characterization Of the Branched Intermediate That Accumulatmentioning

confidence: 99%

“…21 Reversion of the Block G BI to MIP at pH 10 was also observed in the Pyrococcus GB-D Pol intein and is, therefore, a common feature of all classes of inteins. 20 Another difference between the MP-Be DnaB intein and the Dra Snf2 intein was seen in the role of their penultimate His residues. Mutation of the MP-Be DnaB intein penultimate His340 had only a minimal effect on splicing or C-terminal cleavage.…”

Section: Characterization Of the Branched Intermediate That Accumulatmentioning

confidence: 99%

See 1 more Smart Citation

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Brace¹,

Southworth²,

Tori³

et al. 2010

Protein Science

Self Cite

View full text Add to dashboard Cite

Inteins are the protein equivalent of introns. They are remarkable and robust single turnover enzymes that splice out of precursor proteins during post-translational maturation of the host protein (extein). The Deinococcus radiodurans Snf2 intein is the second member of the recently discovered Class 3 subfamily of inteins to be characterized. Class 3 inteins have a unique sequence signature: (a) they start with residues other than the standard Class 1 Cys, Ser or Thr, (b) have a noncontiguous, centrally located Trp/Cys/Thr triplet, and (c) all but one have Ser or Thr at the start of the C-extein instead of the more common Cys. We previously proposed that Class 3 inteins splice by a variation in the standard intein-mediated protein splicing mechanism that includes a novel initiating step leading to the formation of a previously unrecognized branched intermediate. In this mechanism defined with the Class 3 prototypic Mycobacteriophage Bethlehem DnaB intein, the triplet Cys attacks the peptide bond at the N-terminal splice junction to form the class specific branched intermediate after which the N-extein is transferred to the side chain of the Ser, Thr, or Cys at the C-terminal splice junction to form the standard intein branched intermediate. Analysis of the Deinococcus radiodurans Snf2 intein confirms this splicing mechanism. Moreover, the Class 3 specific Block F branched intermediate was isolated, providing the first direct proof of its existence.

show abstract

Section: Characterization Of the Branched Intermediate That Accumulatmentioning

confidence: 99%

Section: Characterization Of the Branched Intermediate That Accumulatmentioning

confidence: 99%

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Brace¹,

Southworth²,

Tori³

et al. 2010

Protein Science

Self Cite

View full text Add to dashboard Cite

show abstract

“…Protein splicing is an autocatalytic process, which does not require any extrinsic protease to assist the excision-coupled ligation reaction, because the purified precursors undergo protein splicing in vitro [13,15]. In the present study, we investigated this unique chemical reaction using an in vitro system.…”

Section: Discussionmentioning

confidence: 99%

Protein splicing in the yeast Vma1 protozyme: evidence for an intramolecular reaction

Kawasaki¹,

Satow²,

Ohya³

et al. 1997

FEBS Letters

View full text Add to dashboard Cite

Protein splicing is an autocatalytic reaction of a single polypeptide in which a spliced intervening sequence is excised out and the two external regions are ligated with the peptide bond to yield two mature proteins. We examined the reaction mechanism using a folding-dependent in vitro protein splicing system. Protein splicing proceeds at an optimal pH of 7 and is an intramolecular reaction. The reaction is not inhibited by potential protease inhibitors, suggesting that its mechanism is different from those catalyzed by known proteases.

show abstract

“…The mechanism of protein splicing was deciphered after establishment of an in vitro assay system (9). This was critical because protein splicing in a native context is usually so fast that precursor is rarely observed.…”

Section: The Standard Protein Splicing Mechanismmentioning

confidence: 99%

Protein Splicing Mechanisms and Applications

Perler

2005

IUBMB Life (International Union of Biochemistry and Molecular B

View full text Add to dashboard Cite

SummaryInteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins). The protein splicing pathway consists of four nucleophilic displacements directed by the intein plus the first C-extein residue. The intein active site(s) are formed by folding of the intein within the precursor, which brings together the splice junctions and internal intein residues that assist catalysis. Inteins with non-canonical catalytic residues splice by modified pathways. Understanding intein proteolytic cleavage and ligation activities has led to the development of many novel applications in the fields of protein engineering, enzymology, microarray production, target detection and activation of transgenes in plants. Recent advances include intein-mediated attachment of proteins to solid supports for microarray or western blot analysis, linking nucleic acids to proteins and controllable splicing, which converts inteins into molecular switches. IUBMB Life, 57: 469 -476, 2005

show abstract

In vitro protein splicing of purified precursor and the identification of a branched intermediate

Cited by 201 publications

References 19 publications

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Protein splicing in the yeast Vma1 protozyme: evidence for an intramolecular reaction

Protein Splicing Mechanisms and Applications

Contact Info

Product

Resources

About