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.

Ligation of multiple protein domains using orthogonal inteins with non-native splice junctions.

Protein splicing is a self-catalyzed process in which an internal protein domain (the intein) is excised from its flanking sequences, linking them together with a canonical peptide bond. Trans-inteins are separated in two different precursor polypeptide chains that must assemble to catalytically self-excise and ligate the corresponding flanking exteins to join even when expressed separately either in vitro or in vivo. They are very interesting to construct full proteins from separate domains because their common small size favors chemical synthesis approaches. Therefore, trans-inteins have multiple applications such as protein modification and purification, structural characterization of protein domains or production of intein-based biosensors, among others. For many of these applications, when using more than one trans-intein, orthogonality between them is a critical issue to ensure the proper ligation of the exteins. Here, we confirm the orthogonality (lack of cross-reactivity) of four different trans- or split inteins, gp41-1, gp41-8, IMPDH-1 and NrdJ-1 both in vivo and in vitro, and built different constructs that allow for the sequential fusion of up to four protein fragments into one final spliced product. We have characterized the splicing efficiency of these constructs. All harbor non-native extein residues at the splice junction between the trans-intein and the neighboring exteins, except for the essential Ser + 1. Our results show that it is possible to ligate four different protein domains using inteins gp41-1, IMPDH-1 and NrdJ-1 with non-native extein residues to obtain a final four-domain spliced product with a not negligible yield that keeps its native sequence.

Novel protein ligase based on dual split intein.

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase.

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 SpyCatcher fused with superfolder GFP in this chapter.

Catcher/Tag Toolbox: Biomolecular Click-Reactions For Protein Engineering Beyond Genetics.

Manipulating protein architectures beyond genetic control has attracted widespread attention. Catcher/Tag systems enable highly specific conjugation of proteins in vivo and in vitro via an isopeptide-bond. They provide efficient, robust, and irreversible strategies for protein conjugation and are simple yet powerful tools for a variety of applications in enzyme industry, vaccines, biomaterials, and cellular applications. Here we summarize recent development of the Catcher/Tag toolbox with a particular emphasis on the design of Catcher/Tag pairs targeted for specific applications. We cover the current limitations of the Catcher/Tag systems and discuss the pH sensitivity of the reactions. Finally, we conclude some of the future directions in the development of this versatile protein conjugation method and envision that improved control over inducing the ligation reaction will further broaden the range of applications.

Inteins-mechanism of protein splicing, emerging regulatory roles, and applications in protein engineering.

Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context, protein splicing results in two separate protein products coupled to the activation of the intein-containing host protein. Inteins are generally described as either full-length inteins, mini-inteins or split inteins, which are differentiated by their genetic structure and features. Inteins can also be divided into three classes based on their splicing mechanisms, which differ in the location of conserved residues that mediate the splicing pathway. Although inteins were once thought to be selfish genetic elements, recent evidence suggests that inteins may confer a genetic advantage to their host cells through posttranslational regulation of their host proteins. Finally, the ability of modified inteins to splice and cleave their fused exteins has enabled many new applications in protein science and synthetic biology. In this review, we briefly cover the mechanisms of protein splicing, evidence for some inteins as environmental sensors, and intein-based applications in protein engineering.

New insights into cancer: MDM2 binds to the citrullinating enzyme PADI4.

PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. MDM2 is an E3 ubiquitin ligase which is crucial for down-regulation of degradation of the tumor suppressor gene p53. Given the relationship between both PADI4 and MDM2 with p53-signaling pathways, we hypothesized they may interact directly, and this interaction could be relevant in the context of cancer. Here, we showed their association in the nucleus and cytosol in several cancer cell lines. Furthermore, binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that MDM2 could bind to the active site of PADI4, as confirmed by in silico experiments. In vitro and in silico studies showed that the isolated N-terminal region of MDM2, N-MDM2, interacted with PADI4, and residues Thr26, Val28, Phe91 and Lys98 were more affected by the presence of the enzyme. Moreover, the dissociation constant between N-MDM2 and PADI4 was comparable to the IC50 of GSK484 from in cellulo experiments. The interaction between MDM2 and PADI4 might imply MDM2 citrullination, with potential therapeutic relevance for improving cancer treatment, due to the generation of new antigens.

Direct Comparison of Lysine versus Site-Specific Protein Surface Immobilization in Single-Molecule Mechanical Assays.

Single-molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force-transducing probes such as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.

Chemical and structural approaches to investigate PTEN function and regulation.

Phosphatase and tensin homolog is a lipid phosphatase that serves as the major negative regulator of the PI3K/AKT pathway. It catalyzes the 3'-specific dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to generate PIP2. PTEN's lipid phosphatase function depends on several domains, including an N-terminal segment spanning the first 24 amino acids, which results in a catalytically impaired enzyme when mutated. Furthermore, PTEN is regulated by a cluster of phosphorylation sites located on its C-terminal tail at Ser380, Thr382, Thr383, and Ser385, which drives its conformation from an open to a closed autoinhibited but stable state. Herein, we discuss the protein chemical strategies we used to reveal the structure and mechanism of how PTEN's terminal regions govern its function.

Protein Ligation-Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E.†coli.

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes.

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins.

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods.

Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1.

NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling. Many examples of segmental isotopic labeling have been described where two segments of a protein are ligated together by chemical or enzymatic means, but there are far fewer descriptions of a three or more segment ligation reaction. Herein, we describe an enzymatic segmental labeling scheme that combines the widely used Sortase A and more recently described OaAEP1 for a two site ligation strategy. In preparation to study proposed long-range allostery in the 104 kDa DNA damage repair protein Rad50, we ligated side-chain methyl group labeled Zn Hook domain between two long segments of otherwise unlabeled P.furiosus Rad50. Enzymatic activity data demonstrated that the scars resulting from the ligation reactions did not affect Rad50 function within the Mre11-Rad50 DNA double strand break repair complex. Finally, methyl-based NMR spectroscopy confirmed the formation of the full-length ligated protein. Our strategy highlights the strengths of OaAEP1 for segmental labeling, namely faster reaction times and a smaller recognition sequence, and provides a straightforward template for using these two enzymes in multisite segmental labeling reactions.

Efficient Multiple Domain Ligation for Proteins Using Asparaginyl Endopeptidase by Selection of Appropriate Ligation Sites Based on Steric Hindrance.

Three domain fragments of a multi-domain protein, ER-60, were ligated in two short linker regions using asparaginyl endopeptidase not involving denaturation. To identify appropriate ligation sites, by selecting several potential ligation sites with fewer mutations around two short linker regions, their ligation efficiencies and the functions of the ligated ER-60s were examined experimentally. To evaluate the dependence of ligation efficiencies on the ligation sites computationally, steric hinderances around the sites for the ligation were calculated through molecular dynamics simulations. Utilizing the steric hindrance, a site-dependent ligation potential index was introduced as reproducing the experimental ligation efficiency. Referring to this index, the reconstruction of ER-60 was succeeded by the ligation of the three domains for the first time. In addition, the new ligation potential index well-worked for application to other domain ligations. Therefore, the index may serve as a more time-effective tool for multi-site ligations.

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation.

Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA5, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA5. The ligated peptide His-NKC-Cys-DOPA5 was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA5 possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA5 can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances.

TRAIL & EGFR affibody dual-display on a protein nanoparticle synergistically suppresses tumor growth.

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer drug candidate because it selectively binds to the proapoptotic death receptors, which are frequently overexpressed in a wide range of cancer cells, subsequently inducing strong apoptosis in these cells. However, the therapeutic benefit of TRAIL has not been clearly proven, mainly because of its poor pharmacokinetic characteristics and frequent resistance to its application caused by the activation of a survival signal via the EGF/epidermal growth factor receptor (EGFR) signaling pathway. Here, a lumazine synthase protein cage nanoparticle isolated from Aquifex aeolicus (AaLS) was used as a multiple ligand-displaying nanoplatform to display polyvalently both TRAIL and EGFR binding affibody molecules (EGFRAfb) via a SpyTag/SpyCatcher protein-ligation system, to form AaLS/TRAIL/EGFRAfb. The dual-ligand-displaying AaLS/TRAIL/EGFRAfb exhibited a dramatically enhanced cytotoxicity on TRAIL-resistant and EGFR-overexpressing A431 cancer cells in vitro, effectively disrupting the EGF-mediated EGFR survival signaling pathway by blocking EGF/EGFR binding as well as strongly activating both the extrinsic and intrinsic apoptotic pathways synergistically. The AaLS/TRAIL/EGFRAfb selectively targeted A431 cancer cells in vitro and actively reached the tumor sites in vivo. The A431 tumor-bearing mice treated with AaLS/TRAIL/EGFRAfb exhibited a significant suppression of the tumor growth without any significant side effects. Collectively, these findings showed that the AaLS/TRAIL/EGFRAfb could be used as an effective protein-based therapeutic for treating EGFR-positive cancers, which are difficult to manage using mono-therapeutic approaches.

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.

Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK.

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) Nϵ -L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.

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.

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.