Protein Ligation Research Articles

Sortase A-Based Post-translational Modifications on Encapsulin Nanocompartments.

Protein-based encapsulin nanocompartments, known for their well-defined structures and versatile functionalities, present promising opportunities in the fields of biotechnology and nanomedicine. In this investigation, we effectively developed a sortase A-mediated protein ligation system in Escherichia coli to site-specifically attach target proteins to encapsulin, both internally and on its surfaces without any further in vitro steps. We explored the potential applications of fusing sortase enzyme and a protease for post-translational ligation of encapsulin to a green fluorescent protein and anti-CD3 scFv. Our results demonstrated that this system could attach other proteins to the nanoparticles' exterior surfaces without adversely affecting their folding and assembly processes. Additionally, this system enabled the attachment of proteins inside encapsulins which varied shapes and sizes of the nanoparticles due to cargo overload. This research developed an alternative enzymatic ligation method for engineering encapsulin nanoparticles to facilitate the conjugation process.

Evaluation of the cyclic single-chain Fv antibody derived from nivolumab by biophysical analyses and in vitro cell-based bioassay.

Single-chain Fv (scFv) is a recombinant small antibody in which a polypeptide linker connects the variable regions of the light chain (VL) and the heavy chain (VH). The practical use of scFv, however, has been prevented by its tendency to aggregate due to interchain VL-VH interactions. We recently developed a cyclic scFv whose N-terminus and C-terminus were connected by protein ligation techniques. Biophysical comparisons between cyclic and linear scFv have been conducted, but cell biological evaluations remain unexplored. Here we studied the properties of cyclic and linear scFv derived from nivolumab. Biophysical studies revealed that the thermal stability was not changed but that the antigen-binding activity was approximately 3-fold higher as a result of circularization. A cell-based PD-1/PD-L1 interaction inhibitory assay revealed that the biological activity of scFv was markedly higher in the circularized form. In addition, biophysical analysis of scFv proteins incubated in the presence of serum revealed that circularization suppressed the decrease in antigen-binding activity. It could be assumed that circularization of scFv improved stability in the presence of serum, which in turn would suggest the applicability of cyclic scFv as a biopharmaceutical format.

The Transpeptidase Sortase A Binds Nucleic Acids and Mediates Mammalian Cell Labeling.

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

Encouraging Solution to the Problem of Synthesizing Protein α‐Thioester

Comprehensive SummaryExpressed protein ligation (EPL) provides a powerful tool to access large‐size proteins with precise structures. Existing methods for constructing the critical protein thioester for EPL have predominantly relied on the recombinant intein fusion expressed in Escherichia coli (E. coli). Despite its powerful applications, the expression of thioester derived from eukaryotic protein in E. coli inherently suffers from its limited solubility, the inactivity of intein, premature hydrolysis and low yields. To overcome these obstacles, we present herein the facile one‐flask synthesis of inaccessible protein α‐thioester via a SUMO‐protein‐intein (SPI) sandwich model. The utility of SUMO enhances the protein fusion yield and solubility, prevents premature hydrolysis and simplifies the purification process. The inaccessible protein thioester with internal Cys residues can be readily produced and is compatible with the EPL‐desulfurization protocol used to prepare complex proteins, which is otherwise difficult to obtain using traditional methods. Its utility has been highlighted through the synthesis of human granulocyte colony‐stimulating factor (G‐CSF).

Publisher Correction: A modular flow platform for sulfur(VI) fluoride exchange ligation of small molecules, peptides and proteins

Publisher Correction: A modular flow platform for sulfur(VI) fluoride exchange ligation of small molecules, peptides and proteins

Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System.

The SpyCatcher-SpyTag system has become a popular and versatile tool for protein ligation. It is based on a small globular protein (SpyCatcher) that binds to a 13-residue peptide (SpyTag), which subsequently leads to the formation of a covalent isopeptide bond. Thus, the reaction is essentially irreversible. Here, we describe how the SpyCatcher-SpyTag system can be used to label surface-exposed bacterial outer membrane proteins, e.g., for topology mapping or fluorescent time-course experiments. We cover using fluorescence measurements and microscopy to measure labeling efficiency using SpyCatcherfused with superfolderGFPin this chapter.

Approaches to the Full and Partial Chemical Synthesis of Proteins.

Histones are proteins which help to organize DNA. The way in which they function is complex and is partially controlled by post-translational modifications (PTMs). Histone proteins from numerous organisms can be recombinantly produced in bacteria, but many bacterial strains are incapable of installing the variety of PTMs that histones possess. An alternative method of producing histones, which can be used to introduce PTMs, is native chemical ligation (NCL). This chapter provides a general NCL protocol which can be used to produce synthetic, post-translationally modified, histone proteins. The focus is on the NCL procedure itself and not on producing the modified histone protein fragments as there are many different ways in which these can be synthesized, depending on the modification(s) required. The same NCL protocol is also applicable for expressed protein ligation (EPL) with only small modifications to the purification procedure potentially required.

Chemical Synthesis Creates Single Glycoforms of the Ectodomain of Herpes Simplex Virus-1 Glycoprotein D.

Herpes simplex virus-1 (HSV-1) utilizes multiple viral surface glycoproteins to trigger virus entry and fusion. Among these glycoproteins, glycoprotein D (gD) functions as a receptor-binding protein, which makes it an attractive target for the development of vaccines against HSV-1 infection. Several recombinant gD subunit vaccines have been investigated in both preclinical and clinical phases with varying degrees of success. It is fundamentally critical to explore the functions of gD glycans. In light of this, we report an efficient synthetic platform to construct glycosylated gDs bearing homogeneous glycans at N94 and N121. The oligosaccharides were prepared by enzymatic synthesis and conjugated to peptidyl sectors. The glycoproteins were constructed via a combination of 7-(piperazin-1-yl)-2-(methyl)quinolinyl (PPZQ)-assisted expressed protein ligation and β-mercapto amino acid-assisted-desulfurization strategies. Biological studies showed that synthetic gDs exhibited potent in vivo activity in mice.

Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation

Cysteine conjugation is an important tool in protein research and relies on fast, mild and chemoselective reactions. Cysteinyl thiols can either be modified with prefunctionalized electrophiles, or converted into electrophiles themselves for functionalization with selected nucleophiles in an independent step. Here we report a bioconjugation strategy that uses a vinyl thianthrenium salt to transform cysteine into a highly reactive electrophilic episulfonium intermediate in situ, to enable conjugation with a diverse set of bioorthogonal nucleophiles in a single step. The reactivity profile can connect several nucleophiles to biomolecules through a short and stable ethylene linker, ideal for introduction of infrared labels, post-translational modifications or NMR probes. In the absence of reactive exogenous nucleophiles, nucleophilic amino acids can react with the episulfonium intermediate for native peptide stapling and protein–protein ligation. Ready synthetic access to isotopologues of vinyl thianthrenium salts enables applications in quantitative proteomics. Such diverse applications demonstrate the utility of vinyl-thianthrenium-based bioconjugation as a fast, selective and broadly applicable tool for chemical biology.

A modular flow platform for sulfur(VI) fluoride exchange ligation of small molecules, peptides and proteins

A modular flow platform for sulfur(VI) fluoride exchange ligation of small molecules, peptides and proteins

Protein cage nanoparticle-based NK cell-engaging nanodrones (NKeNDs) effectively recruit NK cells to target tumor sites and suppress tumor growth

Natural killer (NK) cells are innate lymphoid cells that play a pivotal role in anticancer immunity, and significant efforts have been made to develop effective NK cell-engagers that recruit and activate NK cells to treat cancers. Here, unprecedented NK cell-engaging nanodrones (NKeNDs) are developed using AaLS protein cage nanoparticles that simultaneously display cancer-targeting ligands (HER2Afb or EGFRAfb) and NK cell-recruiting ligands (aCD16Nb) on the surface of the AaLS through the SpyCatcher/SpyTag protein ligation system. These dual ligand-displaying NKeNDs, HER2 @NKeND and EGFR@NKeND, selectively bind to HER2-overexpressing SK-OV-3 cells and EGFR-overexpressing MDA-MB-468 cells, respectively, as well as human NK cells. The NKeND-mediated physical engagement of human NK cells to the target cancer cells leads to the activation of human NK cells, enabling them to effectively kill the target cancer cells in vitro. In SK-OV-3 tumor-bearing mice, the administration of HER2 @NKeNDs along with human PBMCs facilitates the infiltration of activated human NK cells into the tumor sites, resulting in the suppression of tumor growth without noticeable side effects. Our study demonstrates a novel approach for developing cancer-specific NK cell engagers using protein cage nanoparticles and recombinant cancer cell binders, offering potential for the selective treatment of intractable types of cancers.

Near-Infrared Optogenetic Module for Conditional Protein Splicing

Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.

Mineralocorticoid Receptor Antagonism Prevents Type 2 Familial Partial Lipodystrophy Brown Adipocyte Dysfunction.

Type-2 Familial Partial Lipodystrophy (FPLD2), a rare lipodystrophy caused by LMNA mutations, is characterized by a loss of subcutaneous fat from the trunk and limbs and excess accumulation of adipose tissue in the neck and face. Several studies have reported that the mineralocorticoid receptor (MR) plays an essential role in adipose tissue differentiation and functionality. We previously showed that brown preadipocytes isolated from a FPLD2 patient's neck aberrantly differentiate towards the white lineage. As this condition may be related to MR activation, we suspected altered MR dynamics in FPLD2. Despite cytoplasmic MR localization in control brown adipocytes, retention of MR was observed in FPLD2 brown adipocyte nuclei. Moreover, overexpression of wild-type or mutated prelamin A caused GFP-MR recruitment to the nuclear envelope in HEK293 cells, while drug-induced prelamin A co-localized with endogenous MR in human preadipocytes. Based on in silico analysis and in situ protein ligation assays, we could suggest an interaction between prelamin A and MR, which appears to be inhibited by mineralocorticoid receptor antagonism. Importantly, the MR antagonist spironolactone redirected FPLD2 preadipocyte differentiation towards the brown lineage, avoiding the formation of enlarged and dysmorphic lipid droplets. Finally, beneficial effects on brown adipose tissue activity were observed in an FPLD2 patient undergoing spironolactone treatment. These findings identify MR as a new lamin A interactor and a new player in lamin A-linked lipodystrophies.

Peptiligase, an enzyme for efficient chemo-enzymatic synthesis of aviptadil

Increasing attention to peptides as prospective therapeutics has created a renaissance in searching for new alternatives to the current peptide synthetic approaches as well as their modification. In this context, it is necessary to develop different approaches for peptide ligation. Using enzymes as a novel strategy and powerful tool for the peptide and protein ligation has recently received a lot of attention. We here designed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the synthesis of aviptadil a 28-mer therapeutic peptide with potential therapeutic effects in various medical contexts specially in the treatment of acute respiratory distress syndrome (ARDS) by coupling two peptide segments with four different peptiligase variants in aqueous environments. Our study reveals that peptiligase variants are capable of ligation reaction in 15 min. The overall time of ligation is shorter than those peptides with similar lengths and hinderance to aviptadil which reported for conventional synthesis by full solid-phase peptide synthesis. Yields ranged from 54 % to 76 %.

Construction of IgG–Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart’s chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG–Fab2–type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG–Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG–Fab2 type bsAb will expand the bsAb production method.

An improved installation of 2-hydroxy-4-methoxybenzyl (iHmb) method for chemical protein synthesis

The 2-hydroxy-4-methoxybenzyl (Hmb) backbone modification can prevent amide bond-mediated side-reactions (e.g., aspartimide formation, peptide aggregation) by installing the removable Hmb group into a peptide bond, thus improving the synthesis of long and challenging peptides and proteins. However, its use is largely precluded by the limited Hmb's installation sites. In this report, an improved installation of Hmb (iHmb) method was developed to achieve the flexible installation and the convenient removal of Hmb. The iHmb method involves two critical steps: (1) oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl (Hmab) to give 2-hydroxy-4-methoxy-5-diazonium-benzyl (Hmdab) by combining soamyl nitrite (IAN)/HBF4, and (2) reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol (EDT). The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids (e.g., valine and isoleucine). The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide, the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue. Furthermore, the iHmb method can be utilized to facilitate chemical protein ligation, as exemplified by the synthesis of the single-spanning membrane protein sarcolipin. The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.

Compact and modular bioprobe: Integrating SpyCatcher/SpyTag recombinant proteins with zwitterionic polymer-coated quantum dots

The development of quantum dot (QD)-based modular bioprobe that has a compact size and enable a facile conjugation of various biofunctional groups is in high demand. To address this, we surface engineered QDs with zwitterion polymer ligands to have an inherent compact size and derivatized them sequentially with the recombinant proteins SpyCatcher/SpyTag (SC/ST) to use their protein ligation system. SC/ST spontaneously form one complex through the isopeptide bond between them. SC-conjugated QDs (QD–SC) were used as base building blocks. Then, ST–biomolecules were added for modular biofunctionalization. We synthesized compact sized (∼15 nm) QD–SC–ST–affibody (antibody-mimicking small protein for tumor detection) conjugates, which showed successful cell-receptor targeting. The target cell-receptor could be easily tuned by changing the type of ST-affibody. We also demonstrated that anti-human-chorionic-gonadotropin mouse IgG1 antibodies can be labeled on the QD surface by mixing QD–SC with the ST–MG1Nb (mouse-IgG1-specific protein). The immunoassay performance of the antibody-labeled QDs was evaluated using a pregnancy test kit, displaying equivalent detection sensitivity to a commercially available kit. This study proposed an innovative strategy for QD biofunctionalization in a modular manner, which can be expanded to a diverse range of colloidal particle derivatization.

Affinity-induced covalent protein-protein ligation via the SpyCatcher-SpyTag interaction

Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor. One major source for fermentable sugars is lignocellulose. However, lignocellulosic biomass is difficult to degrade, owing to its inherent structural recalcitrance. Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose. Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions, which are robust but nonetheless non-covalent. The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications. Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes, thus requiring separation of reaction steps, thereby increasing overall process cost. To overcome these challenges, we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction. The resultant cohesin-conjugated dockerin complex was resistant to high temperatures, SDS, and urea while high affinity and specificity of the interacting modular components were maintained. Using this approach, a covalently locked, bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates. The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional, biologically active, covalently locked, affinity-based molecular architectures.

Exploiting Molecular Symmetry to Quantitatively Map the Excited-State Landscape of Iron-Sulfur Clusters.

Cuboidal [Fe4S4] clusters are ubiquitous cofactors in biological redox chemistry. In the [Fe4S4]1+ state, pairwise spin coupling gives rise to six arrangements of the Fe valences ("valence isomers") among the four Fe centers. Because of the magnetic complexity of these systems, it has been challenging to understand how a protein's active site dictates both the arrangement of the valences in the ground state as well as the population of excited-state valence isomers. Here, we show that the ground-state valence isomer landscape can be simplified from a six-level system in an asymmetric protein environment to a two-level system by studying the problem in synthetic [Fe4S4]1+ clusters with solution C3v symmetry. This simplification allows for the energy differences between valence isomers to be quantified (in some cases with a resolution of <0.1 kcal/mol) by simultaneously fitting the VT NMR and solution magnetic moment data. Using this fitting protocol, we map the excited-state landscape for a range of clusters of the form [(SIMes)3Fe4S4-X/L]n, (SIMes = 1,3-dimesityl-imidazol-4,5-dihydro-2-ylidene; n = 0 for anionic, X-type ligands and n = +1 for neutral, L-type ligands) and find that a single ligand substitution can alter the relative ground-state energies of valence isomers by at least 103 cm-1. On this basis, we suggest that one result of "non-canonical" amino acid ligation in Fe-S proteins is the redistribution of the valence electrons in the manifold of thermally populated excited states.