Expressed Protein Ligation Research Articles

Expressed Protein Ligation in Flow.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human β-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.

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.

Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.

The semisynthesis of nucleolar human selenoprotein H.

The human selenoprotein H is the only selenocysteine-containing protein that is located in the cell's nucleolus. In vivo studies have suggested that it plays some role in DNA binding, consumption of reactive oxygen species, and may serve as a safeguard against cancers. However, the protein has never been isolated and, as a result, not yet fully characterized. Here, we used a semi-synthetic approach to obtain the full selenoprotein H with a S43T mutation. Using biolayer interferometry, we also show that the Cys-containing mutant of selenoprotein H is capable of binding DNA with sub-micromolar affinity. Employing state-of-the-art expressed protein ligation (EPL), our devised semi-synthetic approach can be utilized for the production of numerous, hard-to-obtain proteins of biological and therapeutic relevance.

Superfast desulfurization for protein chemical synthesis and modification

Superfast desulfurization for protein chemical synthesis and modification

Tuning reduction potentials of type 1 copper center in azurin by replacing a histidine ligand with its isostructural analogues

Type 1 copper proteins have a conserved ligand set of one cysteine and two histidines, with many proteins, such as azurin, also containing an axial methionine. While the cysteine and methionine in azurin have been replaced with their respective isostructural analogues of unnatural amino acids to reveal their roles in tuning electronic structures and functional properties, such as reduction potentials (E°′), the histidine ligands have not been probed in this way. We herein report the substitution of His117 in azurin with three unnatural isostructural analogues, 5-nitrohistidine(Ntr), thiazolylalanine(SHis) and 1-methylhistidine(MeH) by expressed protein ligation. While UV–vis absorption and electron paramagnetic resonance spectroscopies confirm that isostructural replacement results in minimal structural change in the Cu(II) state, the E°′ of these variants increases with increasing pKa of the δ nitrogens of the imidazole. This counter-intuitive relationship between E°′ of the protein and pKa of the sidechain group suggests additional factors may play a role in tuning E°′.

Substrate Recognition by the Peptidyl-(S)-2-mercaptoglycine Synthase TglHI during 3-Thiaglutamate Biosynthesis.

3-Thiaglutamate is a recently identified amino acid analog originating from cysteine. During its biosynthesis, cysteinyl-tRNA is first enzymatically appended to the C-terminus of TglA, a 50-residue ribosomally translated peptide scaffold. After hydrolytic removal of the tRNA, this cysteine residue undergoes modification on the scaffold before eventual proteolysis of the nascent 3-thiaglutamyl residue to release 3-thiaglutamate and regenerate TglA. One of the modifications of TglACys requires a complex of two polypeptides, TglH and TglI, which uses nonheme iron and O2 to catalyze the removal of the peptidyl-cysteine β-methylene group, oxidation of this Cβ atom to formate, and reattachment of the thiol group to the α carbon. Herein, we use in vitro transcription-coupled translation and expressed protein ligation to characterize the role of the TglA scaffold in TglHI recognition and determine the specificity of TglHI with respect to the C-terminal residues of its substrate TglACys. The results of these experiments establish a synthetically accessible TglACys fragment sufficient for modification by TglHI and identify the l-selenocysteine analog of TglACys, TglASec, as an inhibitor of TglHI. These insights as well as a predicted structure and native mass spectrometry data set the stage for deeper mechanistic investigation of the complex TglHI-catalyzed reaction.

Quinoline-Based Photolabile Protection Strategy Facilitates Efficient Protein Assembly.

Native chemical ligation (NCL) provides a powerful solution to assemble proteins with precise chemical features, which enables a detailed investigation of the protein structure-function relationship. As an extension to NCL, the discovery of desulfurization and expressed protein ligation (EPL) techniques has greatly expanded the efficient access to large or challenging protein sequences via chemical ligations. Despite its superior reliability, the NCL-desulfurization protocol requires orthogonal protection strategies to allow selective desulfurization in the presence of native Cys, which is crucial to its synthetic application. In contrast to traditional thiol protecting groups, photolabile protecting groups (PPGs), which are removed upon irradiation, simplify protein assembly and therefore provide minimal perturbation to the peptide scaffold. However, current PPG strategies are mainly limited to nitro-benzyl derivatives, which are incompatible with NCL-desulfurization. Herein, we present for the first time that quinoline-based PPG for cysteine can facilitate various ligation strategies, including iterative NCL and EPL-desulfurization methods. 7-(Piperazin-1-yl)-2-(methyl)quinolinyl (PPZQ) caging of multiple cysteine residues within the protein sequence can be readily introduced via late-stage modification, while the traceless removal of PPZQ is highly efficient via photolysis in an aqueous buffer. In addition, the PPZQ group is compatible with radical desulfurization. The efficiency of this strategy has been highlighted by the synthesis of γ-synuclein and phosphorylated cystatin-S via one-pot iterative ligation and EPL-desulfurization methods. Besides, successful sextuple protection and deprotection of the expressed Interleukin-34 fragment demonstrate the great potential of this strategy in protein caging/uncaging investigations.

Utilizing a Baculovirus/Insect Cell Expression System and Expressed Protein Ligation (EPL) for Protein Semisynthesis.

Protein semisynthesis has been used for the chemoselective linking of synthetic peptides and recombinant protein fragments to generate complete native proteins in good yield. The ability to site-selectively incorporate multiple post-translational chemical modifications (PTMs) into proteins via this approach shows great potential for enhancing understanding of the molecular basis of protein function and regulation. Protein semisynthesis, however, often requires high expression efficiency of the recombinant protein fragments (i.e., high expression yield and ability to preserve protein biological functions), which can be hard to achieve for some human enzymes when using bacterial expression systems. Here, we describe how to use a baculovirus/insect cell expression system and a protein semisynthesis strategy known as expressed protein ligation (EPL) to produce workable levels of proteins of interest containing site-specific chemical modifications. The protocol provides detailed guidance for generating protein C-terminal thioesters for use with the EPL reaction, performing the EPL reaction, and purifying the protein ligation product. We exemplify the protocols by generating protein kinase Akt1 with site-specific phosphorylations installed into its C-terminal tail, for kinetic kinase assays. We hope these methods will help increase the use of protein semisynthesis for elucidating the post-translational regulation of human enzymes involved in cell signaling. © 2022 Wiley Periodicals LLC Basic Protocol 1: Generation of the N-terminal protein of interest (POI) fragment containing a C-terminal thioester moiety Basic Protocol 2: Expressed protein ligation (EPL) of the protein thioester with a synthetic peptide and purification of the protein ligation product Basic Protocol 3: Semisynthesis and biochemical analysis of site-specifically phosphorylated Akt1.

Applying selenocysteine-mediated expressed protein ligation to prepare the membrane enzyme selenoprotein S.

The intrinsically disordered membrane-bound selenoprotein s (selenos) takes part in the protein quality control pathway, vesicle trafficking, and NF-kB signaling. The reactive selenocysteine (Sec) at the penultimate position is responsible for its enzymatic activity. We report the preparation of the soluble segment as well as the full-length selenos using expressed protein ligation. This chapter discusses the practical considerations of expressed protein ligation using selenopeptides and describes our optimized procedure for the semi-synthesis of selenos.

Protein Synthesis via Activated Cysteine-Directed Protein Ligation.

Proteins with a functionalized C-terminus are critical to synthesizing large proteins via expressed protein ligation. To overcome the limitations of currently available C-terminus functionalization strategies, we established an approach based on a small molecule cyanylating reagent that chemically activates a cysteine in a recombinant protein at its N-side amide for undergoing nucleophilic acyl substitution with amines. We demonstrated the versatility of this approach by successfully synthesizing RNAse H with its RNA hydrolyzing activity restored and in vitro nucleosome build with a C-terminal posttranslational modified histone H2A. This technique will expand the landscape of protein chemical synthesis and its application in new research fields significantly.

Synthesis of O-GlcNAcylated small heat shock proteins.

A protein's structure and function often depend not only on its primary sequence, but also the presence or absence of any number of non-coded posttranslational modifications. Complicating their study is the fact that the physiological consequences of these modifications are context-, protein-, and site-dependent, and there exist no purely biological techniques to unambiguously study their effects. To this end, protein semisynthesis has become an invaluable chemical biology tool to specifically install non-coded or non-native moieties onto proteins in vitro using synthetic and/or recombinant polypeptides. Here, we describe two facets of protein semisynthesis (solid-phase peptide synthesis and expressed protein ligation) and their use in generating site-specifically glycosylated small heat shock proteins for functional studies. The procedures herein require limited specialized equipment, employ mild reaction conditions, and can be extended to myriad other proteins, modifications, and contexts.

Multifaceted Regulation of Akt by Diverse C-Terminal Post-translational Modifications.

Akt is a Ser/Thr protein kinase that regulates cell growth and metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. In addition, dual phosphorylation on Ser477 and Thr479 has been shown to activate Akt. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. Here, we investigate the regulatory effects of phosphorylation of Tyr474 and O-GlcNAcylation of Ser473 on Akt. We use expressed protein ligation as a tool to produce semisynthetic Akt proteins containing phosphoTyr474 and O-GlcNAcSer473 to dissect the enzymatic functions of these PTMs. We find that O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 can also partially increase Akt's kinase activity toward both peptide and protein substrates. Additionally, we performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated versus O-GlcNAcylated Ser473 forms. We observed a high similarity in the protein substrates phosphorylated by phosphoSer473 Akt and O-GlcNAcSer473 Akt. Two Akt substrates identified using microarrays, PPM1H, a protein phosphatase, and NEDD4L, an E3 ubiquitin ligase, were validated in solution-phase assays and cell transfection experiments.

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.

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.

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 Digestion of the -Asp-Cys- Sequence for Preparation of Post-translationally Modified Proteins.

Numerous studies of native proteins have been reported on protein folding in this half century. Recently, post-translationally modified proteins are also focused on protein folding. However, it is still difficult to prepare such types of proteins because it requires not only the chemical but also the recombinant techniques. Native chemical ligation (NCL) is a powerful technique for producing target proteins when combined with recombinant techniques, such as expressed protein ligation (EPL). NCL basically requires an N-terminal peptide with a thioester and a C-terminal peptide which should possess a Cys residue at the N-terminus. Numerous efforts have been made to prepare N-terminal peptides carrying a thioester or a derivative thereof. However, a method for preparing C-terminal Cys-peptides with post-translational modifications has not been well developed, making it difficult to prepare such C-terminal Cys-peptides, except for chemical syntheses or enzymatic digestion. We report here on the development of a convenient technique that involves acid hydrolysis at the -Asp-Cys- sequence, to effectively obtain a C-terminal peptide fragment that can be used for any protein synthesis when combined with EPL, even under denatured conditions. Thus, this chemical digestion strategy permits the NCL strategy to be dramatically accelerated for protein syntheses in which post-translational modifications, such as glycosylation, phosphorylation, etc. are involved. In addition, this method should be useful to prepare the post-translationally modified proteins for protein folding.