Protein Ligation Research Articles

Protease-Triggered Release of Stabilized CXCL12 from Coated Scaffolds in an Ex Vivo Wound Model.

Biomaterials are designed to improve impaired healing of injured tissue. To accomplish better cell integration, we suggest to coat biomaterial surfaces with bio-functional proteins. Here, a mussel-derived surface-binding peptide is used and coupled to CXCL12 (stromal cell-derived factor 1α), a chemokine that activates CXCR4 and consequently recruits tissue-specific stem and progenitor cells. CXCL12 variants with either non-releasable or protease-mediated-release properties were designed and compared. Whereas CXCL12 was stabilized at the N-terminus for protease resistance, a C-terminal linker was designed that allowed for specific cleavage-mediated release by matrix metalloproteinase 9 and 2, since both enzymes are frequently found in wound fluid. These surface adhesive CXCL12 derivatives were produced by expressed protein ligation. Functionality of the modified chemokines was assessed by inositol phosphate accumulation and cell migration assays. Increased migration of keratinocytes and primary mesenchymal stem cells was demonstrated. Immobilization and release were studied for bioresorbable PCL-co-LC scaffolds, and accelerated wound closure was demonstrated in an ex vivo wound healing assay on porcine skin grafts. After 24 h, a significantly improved CXCL12-specific growth stimulation of the epithelial tips was already observed. The presented data display a successful application of protein-coated biomaterials for skin regeneration.

Conjugation of a Blood Brain Barrier Peptide Shuttle to an Fc Domain for Brain Delivery of Therapeutic Biomolecules.

The frequency of brain disease has increased significantly in the past years. After diagnosis, therapeutic options are usually limited, which demands the development of innovative therapeutic strategies. The use of antibody–drug conjugates (ADCs) is promising but highly limited by the existence of the blood–brain barrier (BBB). To overcome the impermeability of this barrier, antibody fragments can be engineered and conjugated to BBB peptide shuttles (BBBpS), which are capable of brain penetration. Herein, we linked the highly efficient BBBpS, PepH3, to the IgG fragment crystallizable (Fc) domain using the streamlined expressed protein ligation (SEPL) method. With this strategy, we obtained an Fc-PepH3 scaffold that can carry different payloads. Fc-PepH3 was shown to be nontoxic, capable of crossing an in vitro cellular BBB model, and able to bind to the neonatal Fc receptor (FcRn), which is responsible for antibody long half-life (t1/2). Overall, we demonstrated the potential of Fc-PepH3 as a versatile platform readily adaptable to diverse drugs of therapeutic value to treat different brain conditions.

Cell-Permeable Nanobodies Allow Dual-Color Super-Resolution Microscopy in Untransfected Living Cells.

Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.

Traceless enzymatic protein synthesis without ligation sites constraint.

Protein synthesis and semisynthesis offer immense promise for life sciences and have impacted pharmaceutical innovation. The absence of a generally applicable method for traceless peptide conjugation with a flexible choice of junction sites remains a bottleneck for accessing many important synthetic targets, however. Here we introduce the PALME (protein activation and ligation with multiple enzymes) platform designed for sequence-unconstrained synthesis and modification of biomacromolecules. The upstream activating modules accept and process easily accessible synthetic peptides and recombinant proteins, avoiding the challenges associated with preparation and manipulation of activated peptide substrates. Cooperatively, the downstream coupling module provides comprehensive solutions for sequential peptide condensation, cyclization and protein N/C-terminal or internal functionalization. The practical utility of this methodology is demonstrated by synthesizing a series of bioactive targets ranging from pharmaceutical ingredients to synthetically challenging proteins. The modular PALME platform exhibits unprecedentedly broad accessibility for traceless protein synthesis and functionalization, and holds enormous potential to extend the scope of protein chemistry and synthetic biology.

Load and Display: Engineering Encapsulin as a Modular Nanoplatform for Protein-Cargo Encapsulation and Protein-Ligand Decoration Using Split Intein and SpyTag/SpyCatcher.

Protein cage nanoparticles have a unique spherical hollow structure that provides a modifiable interior space and an exterior surface. For full application, it is desirable to utilize both the interior space and the exterior surface simultaneously with two different functionalities in a well-combined way. Here, we genetically engineered encapsulin protein cage nanoparticles (Encap) as modular nanoplatforms by introducing a split-C-intein (IntC) fragment and SpyTag into the interior and exterior surfaces, respectively. A complementary split-N-intein (IntN) was fused to various protein cargoes, such as NanoLuc luciferase (Nluc), enhanced green fluorescent protein (eGFP), and Nluc-miniSOG, individually, which led to their successful encapsulation into Encaps to form Cargo@Encap through split intein-mediated protein ligation during protein coexpression and cage assembly in bacteria. Conversely, the SpyCatcher protein was fused to various protein ligands, such as a glutathione binder (GST-SC), dimerizing ligands (FKBP12-SC and FRB-SC), and a cancer-targeting affibody (SC-EGFRAfb); subsequently, they were displayed on Cargo@Encaps through SpyTag/SpyCatcher ligation to form Cargo@Encap/Ligands in a mix-and-match manner. Nluc@Encap/glutathione-S-transferase (GST) was effectively immobilized on glutathione (GSH)-coated solid supports exhibiting repetitive and long-term usage of the encapsulated luciferases. We also established luciferase-embedded layer-by-layer (LbL) nanostructures by alternately depositing Nluc@Encap/FKBP12 and Nluc@Encap/FRB in the presence of rapamycin and applied enhanced green fluorescent protein (eGFP)@Encap/EGFRAfb as a target-specific fluorescent imaging probe to visualize specific cancer cells selectively. Modular functionalization of the interior space and the exterior surface of a protein cage nanoparticle may offer the opportunity to develop new protein-based nanostructured devices and nanomedical tools.

Biosynthetic Strategies for Macrocyclic Peptides.

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.

Preparation of Peptide Selenoesters from Their Corresponding Acyl Hydrazides†

Main observation and conclusionSelenoesters are useful substitutes for traditional thioesters in protein ligation chemistry due to their high reactivity in the trans‐thio/selenoesterification reaction. However, existing synthetic routes to access peptide selenoester require a selenoesterification reaction between a selenide and a protected peptide with a free carboxylate at the C‐terminus. Herein, we introduce an efficient method to convert peptide acyl hydrazide, a convenient thioester surrogate, into the desired selenoester for peptide ligation. Our methodology can be applied to fully de‐protected peptides with various C‐terminal amino acid residues in high yield without racemization. We believe that this method provides a useful alternative to access peptide C‐terminal selenoesters for protein ligation.

Development of a yeast cell surface display method using the SpyTag/SpyCatcher system

Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.

Engineering of a Peptide α-N-Methyltransferase to Methylate Non-Proteinogenic Amino Acids.

Introduction of α-N-methylated non-proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre-methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α-N-automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C-terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non-proteinogenic amino acids has not been addressed. An engineered OphMA, intein-mediated protein ligation and solid-phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non-natural amides. This approach may have application in the biotechnological production of therapeutic peptides.

Engineering of a Peptide α-N-Methyltransferase to Methylate Non-Proteinogenic Amino Acids.

Introduction of α‐N‐methylated non‐proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre‐methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α‐N‐automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C‐terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non‐proteinogenic amino acids has not been addressed. An engineered OphMA, intein‐mediated protein ligation and solid‐phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non‐natural amides. This approach may have application in the biotechnological production of therapeutic peptides.

Nanobody Libraries Using Protein Ligation

Introduction Nanobodies (Nb) are single-domain antibodies derived from camelids and are one-tenth the size of normal antibodies. Various methods have been developed to obtain potent Nb using phage display and yeast display, but the processes of introducing mutations and evaluating Nb mutants are separated, making Nb acquisition a relatively time-consuming and costly process. Recently, in vivo continuous evolution, in which genes are diversified within an organism, has been focused on as a means to accelerate the improvement of protein function. In in vivo continuous evolution, mutagenesis enzymes accumulate mutations in the target gene, and the target gene is continuously diversified simply by culturing the organism. Although in vivo continuous evolution is useful, it is not compatible enough with display technologies. For example, in yeast display, Nb is expressed as a fusion protein with a scaffold protein to be immobilized on the yeast cell wall. If the scaffold protein is mutated, the display itself does not occur. In this study, we have developed a method for binding scaffold proteins expressed from different gene cassettes to Nb in yeast and presenting them on the yeast surface using SpyTag/SpyCatcher [1] protein ligation, which irreversibly forms isopeptide bonds. This method can be integrated with in vivo continuous evolution because Nb can be expressed separately from the scaffold proteins required for the surface display system, allowing selective directed evolution of Nb alone. Methods As a model, we generated a yeast strain which produced anti-lysozyme Nb fused with SpyTag (ST, 16-residue) and a 649-stalk scaffold protein fused with an epitope tag and SpyCatcher (SC, 113-residue). To investigate potential crosstalk between yeast cells, two yeast strains, which produced different Nbs and epitope tag, were co-cultured and stained with anti-epitope antibody and fluorescently labeled lysozyme for microscopic observation. To confirm that our display method can be used for library screening, we attempted to enrich the target yeast cells using a flow cytometer. We generated a mixture sample of yeast cells displaying anti-lysozyme Nb or Nb that does not bind lysozyme. The sample was mixed with fluorescently labeled lysozyme, and fluorescence-positive cells were sorted. Results and Discussion Yeast cells producing anti-lysozyme Nb fused with SpyTag (ST) and 649-stalk scaffold protein fused with an epitope tag and SpyCatcher (SC) were stained with anti-HA antibody and fluorescently labeled lysozyme, and we confirmed that 93% of the yeast cells successfully displayed anti-lysozyme Nb on the yeast cell surface. We observed no crosstalk between yeast cells. In the enrichment experiment, we successfully confirmed that the fluorescence-positive cells were enriched to about 94% by using a flow cytometer. In conclusion, our technique is expected to be an efficient cell surface display platform which can be integrated with in vivo continuous evolution[2]. [1] Anthony. et al. PNAS, Dec 2019, 116, 26523. [2] Kajiwara et al. submitted.

A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology.

Sequence-Selective Decapeptide Synthesis by the Parallel Operation of Two Artificial Molecular Machines

We report on the preparation of a decapeptide through the parallel operation of two rotaxane-based molecular machines. The synthesis proceeds in four stages: (1) simultaneous operation of two molecular peptide synthesizers in the same reaction vessel; (2) selective residue activation of short-oligomer intermediates; (3) ligation; (4) product release. Key features of the machine design include the following: (a) selective transformation of a thioproline building block to a cysteine (once it has been incorporated into a hexapeptide intermediate by one molecular machine); (b) a macrocycle-peptide hydrazine linkage (as part of the second machine) to differentiate the intermediates and enable their directional ligation; and (c) incorporation of a Glu residue in the assembly module of one machine to enable release of the final product while simultaneously removing part of the assembly machinery from the product. The two molecular machines participate in the synthesis of a product that is beyond the capability of individual small-molecule machines, in a manner reminiscent of the ligation and post-translational modification of proteins in biology.

Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases.

Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.

Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging.

Photochemistry provides a wide range of alternative reagents that hold potential for use in bimolecular functionalisation of proteins. Here, we report the synthesis and characterisation of metal ion binding chelates derivatised with disubstituted tetrazoles for the photoradiochemical labelling of monoclonal antibodies (mAbs). The photophysical properties of tetrazoles featuring extended aromatic systems and auxochromic substituents to tune excitation toward longer wavelengths (365 and 395 nm) were studied. Two photoactivatable chelates based on desferrioxamine B (DFO) and the aza-macrocycle NODAGA were functionalised with a tetrazole and developed for protein labelling with 89 Zr, 64 Cu and 68 Ga radionuclides. DFO-tetrazole (1) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with 89 Zr. Radiochemical studies and cellular-based binding assays demonstrated that the radiotracer remained stable in vitro retained high immunoreactivity. Positron emission tomography (PET) imaging and biodistribution studies were used to measure the tumour specific uptake and pharmacokinetic profile in mice bearing SK-OV-3 xenografts. Experiments demonstrate that tetrazole-based photochemistry is a viable approach for the light-induced synthesis of PET radiotracers.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from invitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E.coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.

A modular approach to enzymatic ligation of peptides and proteins with oligonucleotides.

Joining peptides and oligonucleotides offers potential benefits, but current methods remain laborious. Here we present a novel approach towards enzymatic ligation of the two modalities through the development of tag phosphoramidites as adaptors that can be readily incorporated onto oligonucleotides. This simple and highly efficient approach paves the way towards streamlined development and production of peptide/protein-oligonucleotide conjugates.

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

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 synthetic and genetically encoded peptides via protein trans-splicing and is followed by intramolecular cyclization through an expressed protein ligation step mediated with a cis-intein. We identified a suitable pair of orthogonal inteins and optimized the conditions for a one-pot cyclization protocol. We report the semisynthesis of model macrocyles with various ring sizes and of the natural product sunflower trypsin inhibitor (SFTI) along with its ornithine analog.

Chemical Synthesis of PDZ Domains.

Developments in chemical protein synthesis have enabled the generation of tailor-made proteins including incorporation of many types of modifications into proteins, enhancing our ability to control site-specificity of protein posttranslational modifications (PTMs), modify protein backbones and introduce photocrosslinking probes. For PDZ (postsynaptic density protein, disks large, zonula occludens) protein domains, expressed protein ligation (EPL) has been employed to introduce analogs of cognate amino acids, amide-to-ester bond mutations, and phosphorylations in the study of PDZ domain-mediated protein-protein interactions (PPIs). Here, we present protocols for EPL of PDZ domains focusing on phosphorylation and amide-to-ester modifications in the PDZ domain proteins.

Covalent and non-covalent albumin binding of Au(i) bis-NHCs via post-synthetic amide modification.

Recent decades have witnessed the emergence of Au(i) bis-N-heterocyclic carbenes (NHCs) as potential anticancer agents. However, these systems exhibit little interaction with serum proteins (e.g., human serum albumin), which presumably impacts their pharmacokinetic profile and tumor exposure. Anticancer drugs bound to human serum albumin (HSA) often benefit from significant advantages, including longer circulatory half-lives, tumor targeted delivery, and easier administration relative to the drug alone. In this work, we present Au(i) bis-NHCs complexes, 7 and 9, capable of binding to HSA. Complex 7 contains a reactive maleimide moiety for covalent protein conjugation, whereas its congener 9 contains a naphthalimide fluorophore for non-covalent binding. A similar drug motif was used in both cases. Complexes 7 and 9 were prepared from a carboxylic acid functionalized Au(i) bis-NHC (complex 2) using a newly developed post-synthetic amide functionalization protocol that allows coupling to both aliphatic and aromatic amines. Analytical, and in vitro techniques were used to confirm protein binding, as well as cellular uptake and antiproliferative activity in A549 human lung cancer cells. The present findings highlight a hitherto unexplored approach to modifying Au(i) bis-NHC drug candidates for protein ligation and serve to showcase the relative benefits of covalent and non-covalent HSA binding.