Semisynthetic head-to-tail cyclized peptides obtained by combining protein trans-splicing and intramolecular expressed protein ligation

2021 ◽  
Author(s):  
Shubhendu Palei ◽  
Henning D. Mootz
Keyword(s):  
Expressed Protein Ligation ◽  
Peptide Bonds ◽  
Protein Ligation ◽  
Native Peptide ◽  
Split Intein ◽  
Trans Splicing ◽  
Macrocyclic Peptides

A dual-intein approach for the preparation of head-to-tail macrocyclic peptides is reported, where synthetic and genetically encoded fragments are ligated by two native peptide bonds. A split intein ligates the...

scholarly journals An Off-the-Shelf Approach for the Production of Fc Fusion Proteins by Protein Trans-Splicing towards Generating a Lectibody In Vitro

2020 ◽  
Vol 21 (11) ◽  
pp. 4011 ◽  
Author(s):  
Anniina Jaakkonen ◽  
Gerrit Volkmann ◽  
Hideo Iwaï
Keyword(s):  
Emerging Infectious Diseases ◽  
Sars Coronavirus ◽  
Protein Ligation ◽  
Binding Domains ◽  
Naturally Occurring ◽  
Split Intein ◽  
Trans Splicing ◽  
Brevibacillus Choshinensis ◽  
Genetic Fusion

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a “lectibody”. Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

scholarly journals Semisynthesis of functional transmembrane proteins in GUVs

2021 ◽  
Author(s):  
K. A. Podolsky ◽  
T. Masubuchi ◽  
G. T. Debelouchina ◽  
E. Hui ◽  
N. K. Devaraj
Keyword(s):  
Cell Communication ◽  
Membrane Interface ◽  
Synthetic Membranes ◽  
Native Peptide ◽  
Split Intein ◽  
Trans Splicing ◽  
Pharmacology Biochemistry ◽  
Membrane Protein Reconstitution ◽  
Cell Death Protein ◽  
Protein Reconstitution

AbstractCellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

scholarly journals Access to site-specific Fc–cRGD peptide conjugates through streamlined expressed protein ligation

2016 ◽  
Vol 14 (40) ◽  
pp. 9549-9553 ◽  
Author(s):  
S. Frutos ◽  
J. B. Jordan ◽  
M. M. Bio ◽  
T. W. Muir ◽  
O. R. Thiel ◽  
...  
Keyword(s):  
Active Site ◽  
Bioactive Peptides ◽  
Expressed Protein Ligation ◽  
Peptide Conjugates ◽  
Site Specific ◽  
Protein Ligation ◽  
Split Intein ◽  
Efficient Route ◽  
Long Acting ◽  
Powerful Strategy

Conjugation of peptides to the Fc fragment of antibodies is a powerful strategy to generate long acting biotherapeutics. We show here an efficient route to obtain fully active, site-specific conjugates of synthetic bioactive peptides using a split intein based approach.

Protein trans-splicing as a protein ligation tool to study protein structure and function

2011 ◽  
Vol 2 (3) ◽  
pp. 183-198 ◽  
Author(s):  
A. Sesilja Aranko ◽  
Gerrit Volkmann
Keyword(s):  
Protein Structure ◽  
Isotope Labeling ◽  
Successful Implementation ◽  
Protein Ligation ◽  
Current State ◽  
Split Intein ◽  
Segmental Isotope Labeling ◽  
Trans Splicing ◽  
And Function ◽  
Protein Reconstitution

AbstractProtein trans-splicing (PTS) exerted by split inteins is a protein ligation reaction which enables overcoming the barriers of conventional heterologous protein production. We provide an overview of the current state-of-the-art in split intein engineering, as well as the achievements of PTS technology in the realm of protein structure-function analyses, including incorporation of natural and artificial protein modifications, controllable protein reconstitution, segmental isotope labeling and protein cyclization. We further discuss factors crucial for the successful implementation of PTS in these protein engineering approaches, and speculate on necessary future endeavours to make PTS a universally applicable protein ligation tool.

scholarly journals Analysis of Protein Kinase Autophosphorylation Using Expressed Protein Ligation and Computational Modeling

2008 ◽  
Vol 130 (17) ◽  
pp. 5667-5669 ◽  
Author(s):  
Kerry A. Pickin ◽  
Sidhartha Chaudhury ◽  
Blair C. R. Dancy ◽  
Jeffrey J. Gray ◽  
Philip A. Cole
Keyword(s):  
Protein Kinase ◽  
Computational Modeling ◽  
Expressed Protein Ligation ◽  
Protein Ligation
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