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Molekuliarnaia Biologiia 2021When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin...
When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be detrimental. In the study, the streptavidin monomer fragment (S1-116) was fused with the intein N-terminal (termed precursor S1-116-IN), and S1-116-IN was expressed in E. coli (BL21). Meanwhile, the SA117-160 fragment was fused with the C-terminal intein, and then this chimeric polypeptide was expressed on magnetosomes by fusion with magnetosome membrance protein MamF. In the in vitro protein splicing system, the purified engineered magnetosomes (BMP-SA117-160-IC) and the S1-116-IN precursor were mixed. Intein-mediated trans-splicing reaction was induced to produce the functional magnetic beads BMP-SA. Our results indicate that intein-mediated protein trans-splicing may lead to efficient synthesis of the recombinant streptavidin on the magnetosomes, showing its promising potential to produce other functional magnetic nanoparticles.
Topics: Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Inteins; Magnetosomes; Protein Splicing; Streptavidin; Trans-Splicing
PubMed: 34837702
DOI: 10.31857/S0026898421060057 -
Chemical Reviews Dec 2002
Review
Topics: Amino Acid Sequence; Biotechnology; Enzyme Precursors; Kinetics; Protein Engineering; Protein Splicing; Recombinant Fusion Proteins
PubMed: 12475209
DOI: 10.1021/cr9601369 -
Cell Jan 1998
Review
Topics: Amino Acid Sequence; Drosophila Proteins; Evolution, Molecular; Hedgehog Proteins; Insect Proteins; Molecular Sequence Data; Protein Precursors; Protein Processing, Post-Translational; Protein Splicing; Sequence Homology, Amino Acid
PubMed: 9489693
DOI: 10.1016/s0092-8674(00)80892-2 -
Current Opinion in Chemical Biology Oct 2001In mammalian cells, protein-protein interactions constitute essential regulatory steps that modulate the activity of signaling pathways. In recent years, several... (Review)
Review
In mammalian cells, protein-protein interactions constitute essential regulatory steps that modulate the activity of signaling pathways. In recent years, several approaches towards understanding the interactions have been developed. We describe herein a new method for detecting protein-protein interactions in vivo based on protein splicing and highlight some potential applications of this technique.
Topics: Animals; Endodeoxyribonucleases; Green Fluorescent Proteins; Humans; Luciferases; Luminescent Proteins; Optics and Photonics; Protein Binding; Protein Splicing; Recombinant Fusion Proteins
PubMed: 11578933
DOI: 10.1016/s1367-5931(00)00244-1 -
Methods in Molecular Biology (Clifton,... 2017Protein trans-splicing is a posttranslational modification that joins two protein fragments together via a peptide a bond in a process that does not require exogenous...
Protein trans-splicing is a posttranslational modification that joins two protein fragments together via a peptide a bond in a process that does not require exogenous cofactors. Towards achieving cellular control, synthetically engineered systems have used a variety of stimuli such as small molecules and light. Recently, split inteins have been engineered to be photoactive by the LOV2 domain (named LOVInC). Herein, we discuss (1) designing of LOV2-activated target proteins (e.g., inteins), (2) selecting feasible splice sites for the extein, and (3) imaging cells that express LOVInC-based target exteins.
Topics: Arabidopsis Proteins; DNA-Binding Proteins; HeLa Cells; Humans; Inteins; Photochemical Processes; Protein Domains; Protein Splicing; Recombinant Fusion Proteins
PubMed: 27714620
DOI: 10.1007/978-1-4939-6451-2_15 -
Laboratory Investigation; a Journal of... Jul 2019In multiple neurodegenerative diseases, including Alzheimer's disease (AD), a prominent pathological feature is the aberrant aggregation and inclusion formation of the... (Review)
Review
In multiple neurodegenerative diseases, including Alzheimer's disease (AD), a prominent pathological feature is the aberrant aggregation and inclusion formation of the microtubule-associated protein tau. Because of the pathological association, these disorders are often referred to as tauopathies. Mutations in the MAPT gene that encodes tau can cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), providing the clearest evidence that tauopathy plays a causal role in neurodegeneration. However, large gaps in our knowledge remain regarding how various FTDP-17-linked tau mutations promote tau aggregation and neurodegeneration, and, more generally, how the tauopathy is linked to neurodegeneration. Herein, we review what is known about how FTDP-17-linked pathogenic MAPT mutations cause disease, with a major focus on the prion-like properties of wild-type and mutant tau proteins. The hypothesized mechanisms by which mutations in the MAPT gene promote tauopathy are quite varied and may not provide definitive insights into how tauopathy arises in the absence of mutation. Further, differences in the ability of tau and mutant tau proteins to support prion-like propagation in various model systems raise questions about the generalizability of this mechanism in various tauopathies. Notably, understanding the mechanisms of tauopathy induction and spread and tau-induced neurodegeneration has important implications for tau-targeting therapeutics.
Topics: Animals; Humans; Microtubules; Mutation; Parkinsonian Disorders; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Protein Splicing; Tauopathies; tau Proteins
PubMed: 30742061
DOI: 10.1038/s41374-019-0197-x -
Molecular BioSystems Apr 2014Protein splicing technology harnesses the ability of inteins to ligate protein fragments, forming a mature protein. This report describes our effort to engineer...
Protein splicing technology harnesses the ability of inteins to ligate protein fragments, forming a mature protein. This report describes our effort to engineer rapamycin-dependent protein splicing of a ribotoxin, called α-sarcin. Engineering this system required the investigation of important splicing parameters, including extein context and splicing temperature. We show α-sarcin splicing is dependent on rapamycin, is inducible with rapid kinetics, and triggers apoptosis in HeLa cells. These findings establish a proof-of-concept for a conditional cell ablation strategy.
Topics: Apoptosis; Cell Line, Tumor; Endoribonucleases; Fungal Proteins; Green Fluorescent Proteins; HeLa Cells; Humans; Inteins; Protein Engineering; Protein Folding; Protein Splicing; Sirolimus
PubMed: 24481070
DOI: 10.1039/c3mb70387h -
Biochimie Jun 2021Inteins are intervening polypeptides that interrupt the functional domains of several important proteins across the three domains of life. Inteins excise themselves from... (Review)
Review
Inteins are intervening polypeptides that interrupt the functional domains of several important proteins across the three domains of life. Inteins excise themselves from the precursor protein, ligating concomitant extein residues in a process called protein splicing. Post-translational auto-removal of inteins remain critical for the generation of active proteins. The perspective of inteins in science is a robust field of research, however fundamental studies centralized upon splicing regulatory mechanism are imperative for addressing more intricate issues. Controlled engineering of intein splicing has many applications; intein inhibition can facilitate novel drug design, while activation of intein splicing is exploited in protein purification. This paper provides a comprehensive review of the past and recent advances in the splicing regulation via metal-intein interaction. We compare the behavior of different metal ions on diverse intein systems. Though metals such as Zn, Cu, Pt, Cd, Co, Ni exhibit intein inhibitory effect heterogeneously on different inteins, divalent metal ions such as Ca and Mg fail to do so. The observed diversity in the metal-intein interaction arises mostly due to intein polymorphism and variations in atomic structure of metals. A mechanistic understanding of intein regulation by metals in native as well as synthetically engineered intein systems may yield potent intein inhibitors via direct or indirect approach.
Topics: Inteins; Metals; Protein Splicing
PubMed: 33727137
DOI: 10.1016/j.biochi.2021.03.006 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Mar 2003Protein splicing is a newly discovered posttranslational editing process that removes an internal protein fragment from the protein precursor. During the splicing... (Review)
Review
Protein splicing is a newly discovered posttranslational editing process that removes an internal protein fragment from the protein precursor. During the splicing process the internal protein fragment, intein, triggered the self-excision from the precursor protein and the concomitant ligation of the flanking protein fragments, exteins. The self-catalysis requires neither auxiliary enzymes nor cofactors and only involves four intramolecular reactions. A number of key catalytic residues in inteins and flanking fragments have been identified, which led to the development of the protein splicing process as a protein engineering tool. Controllable cleavage of the peptide bond at either the N or the C terminus of an intein has allowed the design of novel strategies for manipulation of protein and peptides. Affinity purification of recombinant proteins can be facilitated by fusion the target protein with an intein. The fusion also creates C-terminal thioester, which expands the scope of chemical ligation in protein. Inteins can be engineered in a "split and inverted" configuration to form a cyclic polypeptide consisting of the sequence linking two intein subdomains. This article summarizes the recent advance in the mechanism of protein splicing and its applications in protein purification, protein ligation and protein cyclization.
Topics: Inteins; Peptides, Cyclic; Protein Engineering; Protein Splicing; Proteins
PubMed: 15966332
DOI: No ID Found -
The Journal of Biological Chemistry Jan 2004Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond....
Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.
Topics: Alleles; Alternative Splicing; Amino Acid Sequence; Antibodies, Monoclonal; Aspartic Acid; Bacteria; Catalysis; DNA; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Glutamine; Models, Biological; Molecular Sequence Data; Mutation; Oligonucleotides; Peptides; Protein Binding; Protein Splicing; Protein Structure, Tertiary; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 14593103
DOI: 10.1074/jbc.M311343200