Catalytic Chain Research Articles

Characteristic features of human catalytic light chain, 22F6, showing the suppression of influenza virus infection by the different way

We found unique human catalytic light chains in which #4 clone showed a substantial effect on the suppression of infectivity of influenza virus A type (H1N1). Interestingly, 22F6 clone also showed a unique characteristic to prevent from the infection of influenza virus by the quite different way.Generally, in vitro assay, the virus and the antibody was first mixed and then the solution was incubated for one hour with MDCK cells in medium. Then, the plaques were counted for estimating the infection efficacy. In this study, we employed a new method in which only antibody was incubated with MDCK cells for one hour. After that, the medium solution containing the antibody was discarded and the well was washed out with the medium. Then the influenza virus dissolved in the medium without an antibody was put into the wells, followed by counting the number of plaques.C179 monoclonal antibody has been used in world widely as a famous neutralizing mAb against influenza virus H1N1 and H2N2 types. For the general in vitro assay, the mAb could completely suppress the infection of the virus against MDCK cells as expected but not in our new method. #4 clone showed the similar result. Surprisingly, 22F6 clone could lower the infectivity of the virus in our method. Namely, the functional mechanism of 22F6 clone is quite different from the neutralizing activity of C179 mAb and the catalytic activity of #4 clone.

Recombinant Expression of Human Enteropeptidase Light Chain in Pichia pastoris

The catalytic light chain of the serine protease human enteropeptidase (enterokinase, hEKLC) was expressed as an active recombinant protein in the yeast Pichia pastoris. EKLC shows high degrees of specificity and selectivity for the amino acid sequences DDDDK~X and DDDDR~X, making it useful for separating fusion domains of recombinant proteins. The amino acid sequence of hEKLC was altered to remove N‐linked glycosylation sites by changing Asn to Gln; similarly, an Arg in a potential kexin protease cleavage site was changed to Gln to preclude proteolysis. The cDNA encoding hEKLC was codon‐optimized for expression in P. pastoris and commercially synthesized. Fusion to α‐mating factor targeted the protein for secretion. Active recombinant hEKLC (rhEKLC) was purified from fermentation medium by liquid chromatography using the enzyme's affinity for soybean trypsin inhibitor (STI). Western blot results confirm the identity of rhEKLC using an antibody to bovine enteropeptidase. Enzymatic activity was characterized via cleavage of the synthetic substrates z‐Lys‐SBzl, GDDDDK‐p‐nitroanilide, and GDDDDR‐p‐nitroanilide. Since the enzyme is secreted in active form, its expression is useful for testing P. pastoris expression media and it has proven useful for activating other proteases expressed as fusion proteins linked via DDDDK sequences. NIH grant R15HL091770.

Charged and hydrophobic surfaces on the a chain of shiga-like toxin 1 recognize the C-terminal domain of ribosomal stalk proteins.

Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A1 chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A1 chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A1 variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A1 chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A1 chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A1 chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs.

On the use of Cob(II)alamin as a spin trap in radical polymerization

Hydroxocobalamin reduced with ascorbic acid acts as a spin trap for free radical polymerization in water. The reaction mechanism is dependent on monomer type. An acrylic olefin such as 2-hydroxyethyl acrylate (HEA) is polymerized at pH 7 and in the presence of a conventional radical source mainly through reversible deactivation by a coupling mechanism. In contrast, a methacrylate like polyethylene glycol methacrylate undergoes catalytic chain transfer oligomerization.

Catalytic chain transfer polymerization for molecular weight control in microemulsion polymerization

Catalytic chain transfer using low-spin cobalt(II) complexes is applied in the microemulsion polymerization of methyl and n-butyl methacrylate. In the presence of a catalytic chain transfer agent the rate of polymerization decreases with an increasing cobalt(II) catalyst loading. The course of the polymerization can be captured by the Morgan-Kaler model extended with radical desorption and aqueous phase termination. Proper molecular weight control (i.e. a molecular weight distribution with a dispersity of ∼2) is achieved, however, proves to be strongly dependent on the balance of cobalt(II) molecules and propagating radicals. The presence of a cobalt(II) catalyst enhances the likelihood of homogeneous nucleation, which decreases the colloidal stability in the methyl methacrylate microemulsion polymerizations. The n-butyl methacrylate microemulsion polymerizations on the other hand, display good colloidal stability and a remarkable increase in the nucleation efficiency.

Terminal functional glycopolymers via a combination of catalytic chain transfer polymerisation (CCTP) followed by three consecutive click reactions

The combination of catalytic chain transfer polymerisation (CCTP) with both thiol-ene and copper catalysed alkyne azide coupling (CuAAC) click chemistry has been employed to give a new route to functional glycopolymers for applications in bioconjugation and biological targeting. Ring opening of poly(glycidyl methacrylate) with sodium azide and subsequent reaction with alkyne functional carbohydrates, as prepared by Fischer glycosylation, has been exploited. This combination of a range of efficient chemistry gives a route to multi gram quantities of glycopolymers avoiding the need for living radical polymerisation chemistry.

The unexpected behaviour of epoxidised macromonomers derived from catalytic chain transfer during ring opening copolymerisation

In our attempts to combine catalytic chain transfer polymerisation (CCTP) with ring-opening polymerisation (ROP), we investigated the homopolymerisation and copolymerisation behaviour of epoxidised CCTP-derived (macro)monomers. Attempts at polymerisation and copolymerisation proved futile. However, some interesting end-functionalised polymers were generated during the attempted copolymerisation reactions. The cationic ROP of epoxidised CCTP-derived (macro)monomers was unsuccessful due to the formation of stable 5-membered rings, incapable of propagation. In a copolymerisation of these epoxides with tetrahydrofuran (THF), however, we observed that the epoxides did not act as a comonomer but rather as an end-capper, providing a functional end group. Most surprising, was the observation that the epoxidised CCTP-derived (macro)monomers significantly catalyse the homopolymerisation of THF.

Controlled oligomerisation of isoprene-towards the synthesis of squalene analogues

Poly(isoprene) oligomers have been synthesised by the use of a range of controlled radical polymerisation techniques, including Catalytic Chain Transfer Polymerisation (CCTP), Reversible Addition Fragmentation Chain Transfer Polymerisation (RAFT), Atom Transfer Radical Polymerisation (ATRP) and Reverse Atom Transfer Radical Polymerisation (RATRP). The key molecular targets were (a) a low number average molecular weight equivalent to that of squalene and (b) a final backbone structure that contains a minimum of 75% of the 1,4 addition form of the isoprene monomer. These structural elements closely match those of the natural squalene which is the cosmetic/health care industry benchmark. CCTP and RAFT were successful in achieving the set targets via solution based polymerisation. The yields of oligomer from these techniques were approximately the same (∼42% from CCTP and ∼47% from RAFT), as were the Mns, (750 and 850 ± 220 g mol−1 respectively versus a GPC squalene target Mn of 570 g mol−1) and polydispersities (1.28 and 1.21 respectively versus a GPC squalene target of 1.02). The backbone structure of the materials produced by these techniques were shown to exceed the 75% 1,4 addition target. Therefore, both of these techniques have the potential to produce the target structures on a larger/commercial scale. However, the choice of initiator and/or RAFT agent was found to have a strong influence on the molecular weight obtained. The ATRP and RATRP techniques proved unable to demonstrate sufficient control over the polymerisation to produce the target oligomers under the reactions conditions applied whilst achieving acceptable yields (>40%).

Polyethylene end functionalization using thia-Michael addition chemistry

Thiol end functionalized polyethylenes (PE-SH, Mn around 1000 g mol−1, Đ < 1.3) were used as nucleophiles in thia-Michael additions with different acrylic molecules. It was found that under commonly used practical conditions the addition to methacrylates was very difficult, whereas addition to acrylates was very efficient. First, block copolymers based on PE and poly(methyl methacrylate) (PMMA) were targeted by reaction of PE-SH with PMMA obtained by catalytic chain transfer polymerization (CCTP). The reaction however failed and detailed model experiments using butanethiol and a dimer of MMA showed that the solubilization temperature of PE-SH was an impediment to the success of the reaction. The lack of reactivity towards PMMA obtained by CCTP and methacrylate functions was advantageously used to react molecules containing both an acrylate and a methacrylate group in the presence of tributyl phosphine (PBu3) to produce methacrylate-type PE macromonomers. The presence of a hydroxyl function on 3-(acryloyloxy)-2-hydroxypropyl methacrylate induced side trans-esterification reactions catalyzed by PBu3. This was overcome by using the hydroxyl free 2-(acryloyloxy) ethyl methacrylate. With the latter, the desired PE macromonomer exhibited a functionality as high as 85%. Alternatively, 2-isocyanatoethyl methacrylate could also be reacted with PE-SH to produce a highly functionalized methacrylate type PE macromonomer (functionality 89%). Eventually, the efficiency of the thia-Michael addition of PE-SH onto poly(ethylene glycol) acrylate (PEG-acrylate) was used to synthesize the PE-b-PEG block copolymer.

Biological surface modification by ‘thiol-ene’ addition of polymers synthesised by catalytic chain transfer polymerisation (CCTP)

Copolymers of oligo(ethylene glycol) methyl ether methacrylates and allyl methacrylate have been synthesised by catalytic chain transfer polymerisation (CCTP) and reacted with α-keratin in human hair via Michael addition to cysteine residues. Further modification of the polymer conjugates was carried out using a fluorescent tag as a model via achieved thiol-ene chemistry.

Effect of TiO2 nanoparticle surface functionalization on protein adsorption, cellular uptake and cytotoxicity: the attachment of PEG comb polymers using catalytic chain transfer and thiol–ene chemistry

A successful modification of titanium dioxide (TiO2) nanoparticles surfaces by a grafting-to polymer technique combining catalytic chain transfer and thiol–ene click chemistry is reported. Vinylic end functional polymers were first prepared by catalytic chain transfer polymerization (CCTP) using oligo(ethylene glycol) methacrylate as a monomer. The presence of vinylic end groups was then exploited to attach the polymers to thiol functionalized TiO2 nanoparticles via thiol–ene Michael nucleophilic reactions. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-infrared (ATR-IR), dynamic light scattering (DLS), and thermogravimetric analyses (TGA) were used to verify the successful modification of the TiO2 surface. The modified TiO2 nanoparticles were stable in cell culture media and formed smaller aggregates when compared to non-surface modified nanoparticles. Cellular toxicity of the hybrid TiO2–polymer particles towards human lung cell lines A549 and H1299 in vitro was evaluated. Results from one-dimensional gel electrophoresis show the presence of polymer layers around the particles affects the adsorption of protein onto the TiO2 surface. The reduction in particle aggregate size and changes to the particle surface chemistry, following polymer grafting, was found to reduce cellular uptake and diminish cytotoxicity for both human lung cell lines tested.

Polysaccharide-stabilized core cross-linked polymer micelle analogues

A novel approach is presented for the synthesis of block-copolymers that resemble the architecture of a core cross-linked micelle. The polymers are synthesized from a combination of catalytic chain transfer polymerization (CCTP), thiol-Michael addition chemistry and reductive amination. A hydrophobic hyperbranched core is synthesized via CCTP of methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA), which affords control over the polymer architecture and the degree of chain end-functionality. The vinyl unsaturations of the hyperbranched polymers are converted in nucleophilic pendant amines by thiol-Michael addition using cysteamine hydrochloride. A polysaccharide shell is grafted onto the hyperbranched core via reductive amination with dextran (DEX). The synthesized poly(MMA-co-EGDMA)-b-DEX polymers possess an amphiphilic character, are colloidally stable and resemble the topology of a core cross-linked micelle. The presented methodology provides a robust, modular, and tuneable approach towards the synthesis of amphiphilic core cross-linked micelle analogues.

Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics

The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.

Catalytic Chain Transfer in Continuous Emulsion Polymerization

AbstractIt is demonstrated that CCT‐mediated emulsion polymerization can successfully be carried out in a PSPC. Proper molecular weight control is achieved with only minor quantities of a cobalt(II) complex, illustrating the robustness and scalability of CCT. Despite the successful implementation of CCT in the PSPC, differences in the course of the polymerization and the final latex properties (in terms of the PSD/MWD) for batchwise and PSPC operation are observed. Backmixing occurs to some extend in the PSPC, which increases the steady state CCTA concentration in the column. Consequently, a higher chain transfer activity is observed than expected based on the corresponding batch experiments, which affects the course of the polymerization and the final latex properties.magnified image

Determination of the Critical Chain Length of Oligomers in Dispersion Polymerization.

The critical chain length (jcrit) in dispersion polymerization is systematically investigated utilizing low molecular weight poly(methyl methacrylate) oligomers synthesized by catalytic chain transfer polymerization. The solubility of these oligomers in methanol/water media of different compositions and at different temperatures has been visually determined. The results show that the solubility of the oligomers increases with increasing methanol fraction and increasing temperature. The constructed solubility map allows for an estimate of jcrit as a function of these important polymerization parameters. Furthermore, it is found that the value of jcrit changes with the concentration of the oligomers in the methanol/water medium, an important consideration for understanding the nucleation stage of a dispersion polymerization. The obtained results have been successfully correlated to earlier data reported on the dispersion polymerization of methyl methacrylate.

Prevention of Aggregation and Autocatalysis for Sustaining Biological Activity of Recombinant BoNT/A-LC Upon Long-Term Storage

Protein aggregation during expression, purification, storage, or transfer into requisite assay buffers hampers the use of proteins for in vitro studies. The formation of these aggregates represents a major obstacle in the study of biological activity and also restricts the spectrum of protein products being available for the biomedical applications. The catalytic light chain of botulinum neurotoxin type A undergoes autocatalysis and aggregation after purification upon long-term storage and freeze-thawing. In present study the conditions for the high level expression and purification of biologically active light chain protein of botulinum neurotoxin were optimized from a synthetic gene. Several co-solvents were screened in order to prevent autocatalysis and aggregation of rBoNT/A-LC. The effect of the co-solvents is studied on endopeptidase activity during long term storage of the recombinant protein. The purified rBoNT/A-LC was also evaluated for its immunogenicity.

Methacrylic Stereoblock Copolymers via the Combination of Catalytic Chain Transfer and Anionic Polymerization

A novel synthetic pathway toward stereoblock copolymers by combining catalytic chain transfer polymerization with anionic polymerization is described. Catalytic chain transfer polymerization (CCTP) has been used to synthesize vinyl-terminated polymers, which, after the Michael addition of α-lithioisopropyl isobutyrate, were used as macroinitiators for the anionic polymerization of methacrylate-type monomers. The resultant polymer consists of a predominantly atactic block (originating from the free radical polymerization) and a more isotactoid or syndiotactoid block (originating from the anionic polymerization).

Efficacy of QCDCR formulated CpG ODN 2007 in Nile tilapia against Streptococcus iniae and identification of upregulated genes

The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P<0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P<0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity.

Catalytic and Biochemical Features of a Monoclonal Antibody Heavy Chain, JN1-2, Raised against a Synthetic Peptide with a Hemagglutinin Molecule of Influenza Virus

It has long been an important issue to produce a catalytic antibody that possesses the ability to lose the infectivity of a bacteria or virus. The monoclonal antibody JN1-2 was generated using a synthetic peptide (TGLRNGITNKVNSVIEKAA) conjugated with human IgG. The peptide sequence includes the conserved region of the hemagglutinin molecule (HA(1) and HA(2) domains), which locates on the envelope of the influenza virus and plays an important role in influenza A virus infection. The monoclonal antibody specifically reacted with the HA2 domain, not only of H2 but also of an H1 strain of the H1N1 subtype (H1 strain). The heavy chain (JN1-2-H) isolated from the parent antibody showed catalytic activity cleaving the above antigenic peptide with very high turnover (kcat = 26 min(-1)), and it could slowly degrade the recombinant HA(2) domain by the catalytic function. Interestingly, the heavy chain exhibited the ability to reduce the infectivity of type A H1N1 but not type B, indicating specificity to type A. This characteristic monoclonal catalytic antibody heavy chain could suppress the infection of the influenza virus in vitro assays.

Polymer Architecture Control in Emulsion Polymerization via Catalytic Chain Transfer Polymerization

Hyperbranched polymers are synthesized via a cobalt(II) mediated emulsion copolymerization of methyl methacrylate and diethylene glycol dimethacrylate. The concentration of divinyl monomer and chain transfer activity have to be balanced in order to prevent gelation of the polymer particles. The physiochemical properties of the cobalt(II) complex, specifically the intrinsic chain transfer activity and the partitioning behavior, play a crucial role in governing the polymer architecture (i.e., branched versus cross-linked). Furthermore, an empirical correlation, based on the chain transfer frequency and the divinyl monomer mol fraction, is presented and validated with the results obtained in emulsion polymerization as well as with available literature data.