Intramolecular Isopeptide Bonds Research Articles

SpyTag/Catcher chemistry induces the formation of active inclusion bodies in E. coli

SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis.

An ester bond underlies the mechanical strength of a pathogen surface protein

Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds. Compared to isopeptide bonds, ester bonds are prone to hydrolysis. It remains elusive whether ester bonds can completely block mechanical extension similarly to isopeptide bonds, or whether ester bonds dissipate mechanical energy by bond rupture. Here, we show that an ester-bond containing stalk domain of Cpe0147 is inextensible even at forces > 2 nN. The ester bond locks the structure to a partially unfolded conformation, in which the ester bond remains largely water inaccessible. This allows the ester bond to withstand considerable mechanical forces and in turn prevent complete protein unfolding. However, the protecting effect might be reduced at non-physiological basic pHs or low calcium concentrations due to destabilizing the protein structures. Inspired by this design principle, we engineer a disulfide mutant resistant to mechanical unfolding under reducing conditions.

Genetics, Structure, and Function of Group A Streptococcal Pili.

Streptococcus pyogenes (Group A Streptococcus; GAS) is an exclusively human pathogen. This bacterial species is responsible for a large variety of infections, ranging from purulent but mostly self-limiting oropharynx/skin diseases to streptococcal sequelae, including glomerulonephritis and rheumatic fever, as well as life-threatening streptococcal toxic-shock syndrome. GAS displays a wide array of surface proteins, with antigenicity of the M protein and pili utilized for M- and T-serotyping, respectively. Since the discovery of GAS pili in 2005, their genetic features, including regulation of expression, and structural features, including assembly mechanisms and protein conformation, as well as their functional role in GAS pathogenesis have been intensively examined. Moreover, their potential as vaccine antigens has been studied in detail. Pilus biogenesis-related genes are located in a discrete section of the GAS genome encoding fibronectin and collagen binding proteins and trypsin-resistant antigens (FCT region). Based on the heterogeneity of genetic composition and DNA sequences, this region is currently classified into nine distinguishable forms. Pili and fibronectin-binding proteins encoded in the FCT region are known to be correlated with infection sites, such as the skin and throat, possibly contributing to tissue tropism. As also found for pili of other Gram-positive bacterial pathogens, GAS pilin proteins polymerize via isopeptide bonds, while intramolecular isopeptide bonds present in the pilin provide increased resistance to degradation by proteases. As supported by findings showing that the main subunit is primarily responsible for T-serotyping antigenicity, pilus functions and gene expression modes are divergent. GAS pili serve as adhesins for tonsillar tissues and keratinocyte cell lines. Of note, a minor subunit is considered to have a harpoon function by which covalent thioester bonds with host ligands are formed. Additionally, GAS pili participate in biofilm formation and evasion of the immune system in a serotype/strain-specific manner. These multiple functions highlight crucial roles of pili during the onset of GAS infection. This review summarizes the current state of the art regarding GAS pili, including a new mode of host-GAS interaction mediated by pili, along with insights into pilus expression in terms of tissue tropism.

Streptococcus pneumoniae Type 1 Pilus - A Multifunctional Tool for Optimized Host Interaction.

Streptococcus pneumoniae represents a major Gram-positive human pathogen causing bacterial pneumonia, otitis media, meningitis, and other invasive diseases. Several pneumococcal isolates show increasing resistance rates against antibacterial agents. A variety of virulence factors promote pneumococcal pathogenicity with varying importance in different stages of host infection. Virulence related hair-like structures (“pili”) are complex, surface located protein arrays supporting proper host interaction. In the last two decades different types of pneumococcal pili have been identified: pilus-1 (P1) and pilus-2 (P2) are formed by the catalytic activity of sortases that covalently assemble secreted polypeptide pilin subunits in a defined order and finally anchor the resulting pilus in the peptidoglycan. Within the long pilus fiber the presence of intramolecular isopeptide bonds confer high stability to the sequentially arranged individual pilins. This mini review will focus on S. pneumoniae TIGR4 P1 molecular architecture, the subunits it builds and provides insights into P1 sortase-mediated assembly. The complex P1 architecture (anchor-/backbone-/tip-subunits) allows the specific interaction with various target structures facilitating different steps of colonization, invasion and spreading within the host. Optimized pilin subunit confirmation supports P1 function under physiological conditions. Finally, aspects of P1- host interplay are summarized, including recent insights into P1 mechanobiology, which have important implications for P1 mediated pathogenesis.

Porphyromonas gingivalis fimbrial protein Mfa5 contains a von Willebrand factor domain and an intramolecular isopeptide

The Gram-negative bacterium Porphyromonas gingivalis is a secondary colonizer of the oral biofilm and is involved in the onset and progression of periodontitis. Its fimbriae, of type-V, are important for attachment to other microorganisms in the biofilm and for adhesion to host cells. The fimbriae are assembled from five proteins encoded by the mfa1 operon, of which Mfa5 is one of the ancillary tip proteins. Here we report the X-ray structure of the N-terminal half of Mfa5, which reveals a von Willebrand factor domain and two IgG-like domains. One of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which is the first such bond observed in a Gram-negative bacterium. These features make Mfa5 structurally more related to streptococcal adhesins than to the other P. gingivalis Mfa proteins. The structure reported here indicates that horizontal gene transfer has occurred among the bacteria within the oral biofilm.

High Force Magnetic Tweezers Reveal That Bacterial Adhesion Pili Act as Megadalton-scale Schock Absorbers

Gram positive pathogens colonize the oral cavity with their adhesive pili. These organelles are exposed to large mechanical perturbations, such as mastication or teeth brushing, which constantly challenge the bacterial adhesion. To survive these events, the pilus proteins contain intramolecular isopeptide bonds that confer them high mechanical stability. In the dental plaque pathogen Actinomyces oris, two strategically located isopeptide bonds in the 2nd and the 3rd domain of the pilus protein FimA prevent the mechanical unfolding of this protein. The only structure that can be stretched under force is the 40 residue sequence trapped between both isopeptide bonds, the isopeptide-delimited loop (IDL) motif. Our previous AFM force spectroscopy experiments unveiled the large forces (>600 pN) required to stretch FimA's IDL; however, the low force resolution of AFM prevented us from studying the IDL folding at low forces. Herein, we develop a high force magnetic tweezers force spectroscopy assay to expose FimA's IDL to forces ranging from 4 to 280 pN, allowing us to cover the IDL (un)folding dynamics with nm and sub-pN resolution. Our approach permits us to detect the IDL extension in a few seconds at forces >200 pN, and to monitor IDL folding at forces <15 pN. After IDL folding, FimA mechanical stability increases along time, suggesting a time-dependent maturation of the interfacial contacts established between FimA's 2nd and 3rd domains. Stretching-relaxation experiments reveal that a pilus composed of 150 FimA subunits would be able to dissipate as heat up to ∼4·105 zJ, acting as a megaDalton-scale shock-absorber. Given the ubiquitous presence of this protein motif among the pili of Gram positive bacteria, IDL pharmaceutical targeting could become a strategy to weaken the adhesion resistance of these pathogens.

Engineering of Group A Streptococcus Isopeptide Bonds into Immunoglobulin-Like Protein Domains.

Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.

Molecular Superglues: Discovery and Engineering Orthogonalization.

Molecular superglues covalently ligate two or more macromolecules together into super stable, covalently linked assemblies. The discovery of intramolecular isopeptide and ester bond crosslinks in bacterial adhesin proteins, inspired the development of two distinct protein ligating technologies based on split protein domains. These chemically distinct technologies could be combined as orthogonal (non-cross-reacting) technologies to make complex assemblies. Here we provide simple practical instructions in the discovery, characterisation, and application of orthogonal ester bond crosslinks as molecular superglues. A large toolkit of diverse, orthogonal molecular superglues will expand our assembly repertoire, and afford increasingly more complex one-, two-, and three-dimensional protein nanomaterials with exquisite control over the final molecular architecture.

Molecular Dynamics of Streptococcus pnuenomae and Corynebacterium diphtheriae Pili

Pili are long filamentous protein structures that protrude from bacterial surfaces and increase their virulence. Structurally they are polymers built from monomeric pilin constituents via intermolecular isopeptide bonds by the action of cysteine transpeptidase sortases enzymes. Pili are constructed to withstand hydrodynamic forces of the order of hundreds of piconewtons in the cell to help bacteria attach to surfaces, therefore, it is important to understand the mechanism by which they overcome such external perturbations since this is pertinent to their function as virulence factors. To combat external forces pili are structurally reinforced by additional intramolecular isopeptide bonds which have been shown to be important in their mechanical stability. In this work, we use all atom molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric forms of the pilins of streptococcus pnuenomae (RrgB) and corynebacterium diphtheriae (spaA) in the presence and absence of intramolecular isopeptide bonds. The results from conventional, accelerated and steered molecular dynamics calculations show that the isopeptide bonds restrict pili conformational dynamics and limit structural change in the presence of external forces. Our results agree with the proposal that isopeptide bonds provide mechanical stability and elucidates the molecular basis for this observation.

Probing the potential of CnaB-type domains for the design of tag/catcher systems

Building proteins into larger, post-translational assemblies in a defined and stable way is still a challenging task. A promising approach relies on so-called tag/catcher systems that are fused to the proteins of interest and allow a durable linkage via covalent intermolecular bonds. Tags and catchers are generated by splitting protein domains that contain intramolecular isopeptide or ester bonds that form autocatalytically under physiological conditions. There are already numerous biotechnological and medical applications that demonstrate the usefulness of covalent linkages mediated by these systems. Additional covalent tag/catcher systems would allow creating more complex and ultra-stable protein architectures and networks. Two of the presently available tag/catcher systems were derived from closely related CnaB-domains of Streptococcus pyogenes and Streptococcus dysgalactiae proteins. However, it is unclear whether domain splitting is generally tolerated within the CnaB-family or only by a small subset of these domains. To address this point, we have selected a set of four CnaB domains of low sequence similarity and characterized the resulting tag/catcher systems by computational and experimental methods. Experimental testing for intermolecular isopeptide bond formation demonstrated two of the four systems to be functional. For these two systems length and sequence variations of the peptide tags were investigated revealing only a relatively small effect on the efficiency of the reaction. Our study suggests that splitting into tag and catcher moieties is tolerated by a significant portion of the naturally occurring CnaB-domains, thus providing a large reservoir for the design of novel tag/catcher systems.

Equilibrium Molecular Dynamics of the Monomer and Dimer Units of Streptococcus Pnuemonae and Corynebacterium Diphtheriae Pili

Pili are elongated protein structures that enhance the adhesive abilities and virulence of bacteria. As a result, it is important to understand their structure and mechanical properties. Pili are constructed by joining monomeric units (or pilins) into polymeric fibers via intermolecular isopeptide bonds. Interestingly, the pilins additionally possess intramolecular isopeptide bonds that are formed between the sidechains of lysine and asparagine residues in the same domain of the protein, which are believed to impart mechanical stability to the pili. In this work, we use all atom molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric forms of the pilins of streptococcus pnuemonae (RrgB) and corynebacterium diphtheriae (spaA) in the presence and absence of intramolecular isopeptide bonds. The results reveal how the energetics and dynamics of these subunits are affected by intramolecular isopeptide bonds, and demonstrate how they affect pili stability.

The crystal structure of the ligand-binding region of serine-glutamate repeat containing protein A (SgrA) of Enterococcus faecium reveals a new protein fold: functional characterization and insights into its adhesion function.

Antimicrobial-resistant and hospital-adapted Enterococcus faecium represents a clinical problem in immunocompromised patients in hospitals worldwide. Understanding the molecular pathogenesis of E. faecium infections may provide novel therapies to treat or prevent infections. A potential target for novel treatment therapies is the serine-glutamate repeat containing protein A (SgrA), which is a cell wall-anchored LPxTG surface protein implicated in binding to fibrinogen and nidogen. Here, we report the X-ray crystal structure of the N-terminal ligand-binding domain of SgrA (rSgrA28-153 ) to a resolution of 1.79 Å. The structure revealed a new protein fold with significant differences from previously characterized DEv-IgG- and inv-IgG-like folds of adhesive proteins known as microbial surface components recognizing adhesive matrix molecules. The structure contains a Lys-Asn-Glu triad with the potential to form a Lys-Asn isopeptide bond. However, even in the absence of a stabilizing intramolecular isopeptide bond, rSgrA28-153 exhibits remarkable properties like resistance to proteases and high thermal stability. The interaction of rSgrA28-153 with fibrinogen, nidogen, laminin, and abiotic surfaces has been characterized and rSgrA28-153 binds to these molecules with high affinity. rSgrA28-153 also binds to the beta chain of fibrinogen and with high affinity and specificity. Strikingly, the presence of 29 surface-exposed hydrophobic amino acid residues likely play an important role in the selectivity of rSgrA28-153 to bind to different abiotic surfaces. The results obtained from this study have opened new avenues to explore and understand the role of this unique surface adhesin in the pathogenesis of catheter-related infections. The coordinates and structure factors for rSgrA28-153 have been deposited with the accession code 5FCE.

Pilus Assembly in Gram-Positive Bacteria.

Pili of Gram-positive bacteria are unique structures on the bacterial surface, assembled from covalently linked polypeptide subunits. Pilus assembly proceeds by transpeptidation reactions catalyzed by sortases, followed by covalent anchoring of the filament in the peptidoglycan layer. Another distinctive property is the presence of intramolecular isopeptide bonds, conferring extraordinary chemical and mechanical stability to these elongated structures. Besides their function in cell adhesion and biofilm formation, this section discusses possible application of pilus constituents as vaccine components against Gram-positive pathogens.

Surviving a Bumpy Ride in the Oropharynx: Bacterial Pili as Nano-Seatbelts that Dissipate Mechanical Energy

Bacterial pili function in cellular adhesion, and must withstand large mechanical stresses in host environments, such as coughing and chewing. In gram positive bacteria, pili are covalently-linked polymers of single protein subunits, termed pilins. Gram positive pilins uniquely possess intramolecular isopeptide bonds that bridge the peptide backbone to form bypass force transduction pathways. In the crystal structure of Spy0128, a pilin from Streptococcus pyogenes, isopeptide bonds link the N- and C-terminal β-strands. Consequently, Spy0128 is mechanically inextensible. Here we report on the mechanical properties of two related pilins, SpaA from Corynebacterium diphtheriae and FimA from Actinomyces oris, using atomic force microscopy (AFM)-based single molecule force spectroscopy. In the crystal structures of SpaA and FimA, the isopeptide bonds do not directly link the N- and C-terminal β-strands in a single pilin domain. Instead, the isopeptide arrangement creates a ∼40 residue polypeptide loop that resembles a slackened seatbelt, which we predict is sensitive to mechanical unfolding. We find that both SpaA and FimA extend to 14 nm under mechanical force, consistent with our structure-based prediction of unfolding of the nano-seatbelt from a slackened to a taut conformation. At a loading rate of 400 nm/s, these loops unfold at forces of ∼503pN in SpaA and ∼665pN in FimA; as such, SpaA and FimA are among the most mechanically stable proteins yet reported. When the force perturbation is removed, the loops refold at a rapid rate of 29 s−1 or higher. Remarkably, the mechanical stabilities are ∼75pN weaker upon refolding, suggesting that gaining full mechanical stability requires maturation. The high mechanical stability and rapid refolding of the nano-seatbelts suggest a mechanism whereby pilin subunits, polymerized as tens-to-hundreds of repeats in pili, readily absorb and recover from mechanical shocks.

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

Streptococcus pyogenes and other Gram-positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild-type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Isopeptide Bonds Mechanically Stabilize Spy0128 in Bacterial Pili

Pili on the surface of Streptococcus pyogenes play a crucial role in adhesion to and colonization in human cells. The major pilin subunit, Spy0128, features intramolecular covalent isopeptide bonds that autocatalytically form between the side chains of lysine and asparagine residues and are regarded as important factors in conveying structural stability. In support of this notion, single-molecule force spectroscopy experiments with Spy0128 recently demonstrated the inextensibility of these bonds under mechanical load. However, the molecular determinants of their apparent absolute durability remain unknown. Here, we studied the impact of the isopeptide bond in the Spy0128 C-terminal domain on the mechanical properties of this subunit using force-probe molecular dynamics simulations and force distribution analysis. Even in the presence of the covalent cross-link, the pili β-sandwich domain undergoes partial unfolding, albeit at ∼50% higher rupture forces and with the ability to rapidly refold on the nanosecond timescale. We find that the isopeptide bond is located right at the point of stress concentration in the protein, leading to relative, yet not absolute, mechanical stabilization by the additional cross-link. Our findings indicate how the isopeptide bond enhances the mechanical stability and refolding capability at the molecular level, ensuring that the domain remains predominantly in a potentially adhesive conformation.

The Pilin Protein FimP from Actinomyces oris: Crystal Structure and Sequence Analyses

The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.

Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus

Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase-mediated cross-linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA - CNA(1), CNA(2), XNA and CNA(3) - each acquire intramolecular lysine-asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showed that mutants unable to form the intramolecular isopeptide bonds in the CNA(2) or CNA(3) domains retain the ability to form pilus bundles. A mutant lacking the CNA(1) isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X-ray crystallography revealed that the BcpA variant Asp(312) Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly-roll structure of XNA. The Asp(312) Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA(1) and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.

413 Effect of Hydrolysis and Polymerization on Bovine Beta-lactoglobulin Immunoreactivity

BackgroundEnzymatic treatments such as hydrolysis with proteases and polymerization using transglutaminase (TG) have been studied to reduce the immunoreactivity of β-Lactoglobulin (β-Lg). Bromelain is a cisteine protease that is not usually used for production of hypoallergenic hydrolysates. TG is an enzyme that catalyses the formation of inter and intramolecular isopeptide bonds between glutamine and lysine residues. The present study is aimed at investigating the antigenic response of β-Lg polymerized by TG pre or post hydrolysis with bromelain.Methodsβ-Lg (donated by Davisco Inc), was hydrolyzed with bromelain (3% protein w/w in distilled water, 25 U enzyme g−1 of substrate, pH 7.5, 240 min) and then polymerized by TG (7% hydrolysate, 10 or 25 U TG g−1 protein, 50°C/180 min). When polymerization was carried out pre hydrolysis, β-Lg (7% w/w) was polymerized by TG (10 U TG g−1 protein, 50°C/180 min) in 0.1 M Cys or after heat treatment (80°C/60 min), and then hydrolyzed under the same conditions previously described. The hydrolysis reaction was monitored by pH-stat method and the samples were evaluated by SDS-PAGE/tricine. Antigenicity was evaluated by Immunoblotting and ELISA assays using sera from mice sensitized with β-Lg (IgE anti-β-Lg).ResultsThe treatment with TG (10 or 25 U TG g−1), post hydrolysis, lead to formation of products with a wide molecular weight (MW) distribution (3 to 26 kDa), and other products with high MW, indicating partial polymerization. The samples obtained from polymerization pre hydrolysis showed bands with low MM (<6.5 kDa) and also products >26 kDa, indicating a partial hydrolysis of the polymers. Immunoblotting analysis showed no reaction towards specific IgE with any of the samples. ELISA assay showed that the IgE-binding response to sample polymerized by TG post or pre hydrolysis with bromelain were significantly reduced (IgE ≤ 35.21 ug mL−1) as compared to untreated β-Lg (IgE 216.20 ug mL−1 ± 27.58) (P < 0.05).ConclusionsThese results suggested that hydrolysis with bromelain combined with polymerization by TG was capable of reducing the antigenicity of β-Lg, being a potential method for modifying the antigenic properties of proteins.

An Irreversible Lock to Proteins for Dynamic Force Spectroscopy at the Mammalian Cell Surface

Exploring the role of force in biology is often limited by breakage of protein interactions. We have developed a protein interaction that will not break, because a covalent bond forms spontaneously between a genetically-encoded protein (termed SpyCatcher) and a peptide (SpyTag). The covalent bond was formed between engineered fragments of a protein from Streptococcus pyogenes, which naturally forms an intramolecular isopeptide bond. SpyTag could be inserted at various locations in proteins and reacted rapidly and specifically, both in bacterial and mammalian systems.