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Methods in Molecular Biology (Clifton,... 2020Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and... (Review)
Review
Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size. This methodology has been developed based on the knowledge obtained from protein splicing. Protein splicing is a multistep biochemical reaction that includes the concomitant cleavage and formation of peptide bonds carried out by self-processing domains named inteins. The natural substrates of protein splicing are essential proteins found in intein-containing organisms; inteins are also functional in nonnative frameworks and can be used to alter nearly any protein's primary amino acid sequence. Accordingly, different reactivity features of inteins have been largely exploited to manipulate proteins in countless methods encompassing fields from biochemical research to the development of biotechnological applications including the study of disease progression and validation of potential drug candidates. Here, we review almost three decades of research to uncover the chemical and biochemical enigmas of protein splicing and the development of inteins as potent protein engineering tools.
Topics: Biotechnology; Isotope Labeling; Peptides, Cyclic; Protein Engineering; Protein Splicing; Recombinant Proteins
PubMed: 32144661
DOI: 10.1007/978-1-0716-0434-2_2 -
Current Protein & Peptide Science 2019Protein splicing domains, also called inteins, have become a powerful biotechnological tool for applications involving molecular biology and protein engineering. Early... (Review)
Review
Protein splicing domains, also called inteins, have become a powerful biotechnological tool for applications involving molecular biology and protein engineering. Early applications of inteins focused on self-cleaving affinity tags, generation of recombinant polypeptide α-thioesters for the production of semisynthetic proteins and backbone cyclized polypeptides. The discovery of naturallyoccurring split-inteins has allowed the development of novel approaches for the selective modification of proteins both in vitro and in vivo. This review gives a general introduction to protein splicing with a focus on their role in expanding the applications of intein-based technologies in protein engineering and chemical biology.
Topics: Binding Sites; Biocatalysis; Biosensing Techniques; Biotechnology; Inteins; Peptides; Protein Binding; Protein Conformation; Protein Engineering; Protein Splicing; Proteins; Recombinant Proteins
PubMed: 30734675
DOI: 10.2174/1389203720666190208110416 -
Biological Chemistry Mar 2019Synthetic biologists aim at engineering controllable biological parts such as DNA, RNA and proteins in order to steer biological activities using external inputs.... (Review)
Review
Synthetic biologists aim at engineering controllable biological parts such as DNA, RNA and proteins in order to steer biological activities using external inputs. Proteins can be controlled in several ways, for instance by regulating the expression of their encoding genes with small molecules or light. However, post-translationally modifying pre-existing proteins to regulate their function or localization leads to faster responses. Conditional splicing of internal protein domains, termed inteins, is an attractive methodology for this purpose. Here we discuss methods to control intein activity with a focus on those compatible with applications in living cells.
Topics: Animals; Humans; Inteins; Protein Processing, Post-Translational; Protein Splicing; Proteins
PubMed: 30226200
DOI: 10.1515/hsz-2018-0309 -
Biochemical and Biophysical Research... May 2023Conditional protein splicing is a powerful biotechnological tool that can be used to post-translationally control the activity of target proteins. Here we demonstrated a...
Conditional protein splicing is a powerful biotechnological tool that can be used to post-translationally control the activity of target proteins. Here we demonstrated a novel conditional protein splicing approach in which the small ubiquitin-like modifier (SUMO) protease induced the splicing of an atypical split intein. The engineered Ter DnaE-3 S11 split intein which has a small C-intein segment with only 6 amino acids was used in this study. A SUMO tag was fused to the N-terminus of the C-intein to inhibit the protein trans-splicing in vitro. The splicing products could be detected in 15 min with the addition of SUMO protease by western blotting and the splicing efficiency was ∼4-fold higher than the control without SUMO protease for overnight reaction. This engineered Ter DnaE-3 S11 split intein-mediated protein trans-splicing had been further shown to be triggered by SUMO protease in different exteins in vitro. Our study provides new insights into the regulation of protein splicing and is a promising tool for the control of protein structure and function in vitro.
Topics: Protein Splicing; Peptide Hydrolases; Ubiquitin; Inteins; Proteins; Endopeptidases
PubMed: 36924678
DOI: 10.1016/j.bbrc.2023.03.023 -
The Journal of Biological Chemistry May 2014Inteins are nature's escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host... (Review)
Review
Inteins are nature's escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the splicing steps are controlled.
Topics: Amino Acids; Exteins; Inteins; Models, Genetic; Molecular Structure; Protein Precursors; Protein Splicing; Proteins
PubMed: 24695729
DOI: 10.1074/jbc.R113.540310 -
Current Opinion in Chemical Biology Oct 1997It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However,... (Review)
Review
It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.
Topics: Animals; Humans; Hydrolysis; Protein Splicing
PubMed: 9667864
DOI: 10.1016/s1367-5931(97)80065-8 -
IUBMB Life Jul 2005Inteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins).... (Review)
Review
Inteins 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.
Topics: Animals; Humans; Protein Splicing; Proteins
PubMed: 16081367
DOI: 10.1080/15216540500163343 -
Chemical Record (New York, N.Y.) 2006Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein... (Review)
Review
Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step protein splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor proteins with mutations in catalytic residues, which stabilize the precursors for crystallographic study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were determined using conditional trans-splicing (splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the protein splicing pathway has allowed scientists to harness inteins for numerous applications.
Topics: Inteins; Molecular Structure; Protein Splicing; Proteins; Stereoisomerism
PubMed: 16900466
DOI: 10.1002/tcr.20082 -
Genes To Cells : Devoted To Molecular &... Jun 1997Protein splicing is a chemical reaction in which a spliced intervening polypeptide is excised from a precursor protein and the flanking N- and C-terminal regions are... (Review)
Review
Protein splicing is a chemical reaction in which a spliced intervening polypeptide is excised from a precursor protein and the flanking N- and C-terminal regions are ligated with the peptide bond to produce two mature proteins. This unique autocatalytic reaction was first discovered in the yeast VMA1 protein, a 120kDa spliced polypeptide encoded by the VMA1 gene of Saccharomyces cerevisiae. The VMA1 protein catalyses a self protein splicing post-translationally to yield the 70 kDa catalytic subunit of the vacuolar H+-ATPase and the 50 kDa DNA endonuclease. Accumulating evidence has indicated that splicing precursors distribute widely in many organisms covering eukarya, bacteria and archaea. This article argues and summarizes current chemical and biological views on protein splicing.
Topics: Amino Acid Sequence; Binding Sites; Endodeoxyribonucleases; Molecular Sequence Data; Protein Processing, Post-Translational; Protein Splicing; Proton-Translocating ATPases; Saccharomyces cerevisiae Proteins; Vitamin D
PubMed: 9286854
DOI: 10.1046/j.1365-2443.1997.1270325.x -
Annual Review of Genetics 2000Intein is the protein equivalent of intron and has been discovered in increasing numbers of organisms and host proteins. A self-splicing intein catalyzes its own removal... (Review)
Review
Intein is the protein equivalent of intron and has been discovered in increasing numbers of organisms and host proteins. A self-splicing intein catalyzes its own removal from the host protein through a posttranslational process of protein splicing. A mobile intein displays a site-specific endonuclease activity that confers genetic mobility to the intein through intein homing. Recent findings of intein structure and the mechanism of protein splicing illuminated how inteins work and yielded clues regarding intein's origin, spread, and evolution. Inteins can evolve into new structures and new functions, such as split inteins that do trans-splicing. The structural basis of intein function needs to be identified for a full understanding of the origin and evolution of this marvelous genetic element.
Topics: DNA-Directed DNA Polymerase; Evolution, Molecular; Protein Splicing
PubMed: 11092822
DOI: 10.1146/annurev.genet.34.1.61