Split Inteins

doi:10.1007/978-1-4939-6451-2

Cited by 5 publications

(2 citation statements)

References 5 publications

(5 reference statements)

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“…The inteins that have been so far identified in genomes are either encoded by a single gene or by two separate ones [3] [4]. The two fragments of split inteins (which we will refer to as N-intein and C-intein) spontaneously form a complex and perform a trans -splicing reaction following what has been, at least for the Npu DnaE intein, reported to be a “capture and collapse” mechanism [5] [6]. It is likely that split inteins evolved from a contiguous one after genomic rearrangement(s) that led to the separation of the coding sequence into two parts [4].…”

Section: Introductionmentioning

confidence: 99%

Identification of functionalNpuDnaE and gp41-1 inteins split in three fragments

Weis

Palanisamy

Ballestin

et al. 2023

Preprint

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Inteins are special proteins that auto-catalytically carry out a protein splicing reaction. Due to their ability to post-translationally modify target proteins in vitro and in vivo, they are used in different applications, ranging from protein purification to the construction of Boolean logic gates. So far inteins have been found to be either encoded by a single gene (contiguous inteins) or by two separate ones (split inteins). Previously, it has been shown that the contiguous Ssp and Rma DnaB inteins could be artificially split in three fragments and retain functionality. Here we report the identification of novel split sites within the N-terminal fragments of the ultra-fast and efficient Npu DnaE and gp41-1 split inteins that lead to synthetic functional three-piece versions of these inteins. Our data suggest that splitting inteins in three fragments is generally applicable and pave the way for new biotechnological applications based on highly-fragmented inteins.

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Section: Introductionmentioning

confidence: 99%

Identification of functionalNpuDnaE and gp41-1 inteins split in three fragments

Weis

Palanisamy

Ballestin

et al. 2023

Preprint

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“…Enzyme-mediated ligation provides a site-selective route to achieve stable peptide cyclization, owing to the ability of enzymes to recognize and modify specific peptide motifs [23][24][25]. A wide range of enzymes have been employed to achieve peptide or protein cyclization such as sortase [26,27], butelase 1 [28], the S-adenosylmethionine enzyme AlbA [29], subtiligase [30], the thioesterase domain of tyrocidine synthetase [31], inteins [32][33][34][35], and transglutaminase [24]. Despite their proven potential, only a few examples are reported where enzymes were used to engineer display libraries of cyclic peptides for de novo discovery of bioactive peptides [36][37][38].…”

Section: Introductionmentioning

confidence: 99%

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands

Bowen

Schneible

Bacon

et al. 2021

IJMS

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We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.

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Single-Step Non-Chromatographic Purification of Recombinant Mammalian Proteins Using a Split Intein ELP Tag System

Prabhala,

Wood

2023

Methods in Molecular Biology

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Split Inteins

Cited by 5 publications

References 5 publications

Identification of functionalNpuDnaE and gp41-1 inteins split in three fragments

Identification of functionalNpuDnaE and gp41-1 inteins split in three fragments

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands

Single-Step Non-Chromatographic Purification of Recombinant Mammalian Proteins Using a Split Intein ELP Tag System

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