Protein LG Research Articles

Universal Enzyme-Linked Immunosorbent Assays (ELISA) and Utility in the Detection of Antibodies against Salmonella spp. in Several Animal Species

The aim of this study was to confirm the feasibility of using hybrid immunoglobulin-binding reagents in ELISAs for IgG/IgY detection and detection of specific antibodies against an infectious microorganism (Salmonella spp.) in various animal species using a universal diagnostic ELISA. Hybrid immunoglobulin-binding bacterial proteins (IBP), including recombinant protein LA, recombinant protein LG, and recombinant protein AG, have been produced to improve their binding affinity to a much larger number of immunoglobulins. Thus, this hybrid bacterial protein represents a powerful tool for the binding, detection, and purification of immunoglobulins and their fragments. However, SpLA-LG-peroxidase and SpLAG-anti-IgY-peroxidase were produced using the periodate method. These compounds have been shown to be effective as reagents. Their binding affinity to immunoglobulins surpasses that of previously reported hybrid IgG-binding proteins, including the most known SpAG, SpLA, and SpLG. The IgY fraction was isolated from the egg yolks of various birds, including chicken, bantam hen, guinea hen, quail, goose, duck, wild and domestic pigeons, parakeets, cattle egrets, pheasants, and ostrich. The IgY fraction was isolated using the chloroform-polyethylene glycol (PEG) method. An ELISA for anti-Salmonella spp. antibodies was employed with some modifications to determine the presence of antibodies in humans, laying hens, geese, quails, and pigeons. Salmonella is a motile, flagellated, rod-shaped zoonotic pathogen that can survive in the presence or absence of oxygen. They belong to the family Enterobacteriaceae and are implicated in typhoid fever and food-borne illnesses. This pathogen is associated with several diseases, which may become fatal and negatively impact the health of individuals and various economies globally. The poultry industry is most vulnerable to the influence of this pernicious microbe. The authors concluded that universal enzyme-linked immunosorbent assays were effective and reproducible in detecting immunoglobulins from both avian and mammalian species, but ELISA using the SpLAG-anti-IgY-HRP conjugate only reacted with the whole panel of animal antibodies. This conjugate was further used to standardize a universal ELISA for the determination of anti-Salmonella antibodies, in which human and avian species were serologically assessed.

Involvement of the PKA pathway and inhibition of voltage gated Ca2+ channels in antihyperalgesic activity of Lippia grata/β-cyclodextrin

Neuropathic pain (NP) is a difficult condition to treat because of the modest efficacy of available drugs. New treatments are required. In the study we aimed to investigate the effects of the essential oil from Lippia grata alone or complexed in β-cyclodextrin (LG or LG-βCD) on persistent inflammatory and neuropathic pain in a mouse model. We also investigated Ca2+ currents in rat dorsal root ganglion (DRG) neurons. Male Swiss mice were treated with LG or LG/β-CD (24 mg/kg, i.g.) and their effect was evaluated using an acute inflammatory pleurisy model and nociception triggered by intraplantar injection of an agonist of the TRPs channels. We also tested their effect in chronic pain models: injection of Freund's Complete Adjuvant and partial sciatic nerve ligation (PSNL). In the pleurisy model, LG reduced the number of leukocytes and the levels of TNF-α and IL-1β. It also inhibited cinnamaldehyde and menthol-induced nociceptive behavior. The pain threshold in mechanical and thermal hyperalgesia was increased and paw edema was decreased in models of inflammatory and neuropathic pain. PSNL increased inflammatory protein contents and LG and LG-βCD restored the protein contents of TNF-α, NF-κB, and PKA, but not IL-1β and IL-10. LG inhibited voltage gated Ca2+ channels from DRG neurons. Our results suggested that LG or LG-βCD produce anti-hyperalgesic effect in chronic pain models through reductions in TNF-α levels and PKA, and inhibited voltage-gated calcium channels and may be innovative therapeutic agents for the management of NP.

Magnetic Oleosome as a Functional Lipophilic Drug Carrier for Cancer Therapy.

In the present study, we fabricated magnetic oleosomes functionalized with recombinant proteins as a new carrier for oil-based lipophilic drugs for cancer treatment. The bioengineered oleosome is composed of neutral lipids surrounded by a phospholipid monolayer with embedded oleosin fusion proteins. The oleosin was genetically fused to a nanobody of a green fluorescent protein (GFP). A recombinant protein consisting of immunoglobulin-binding protein LG fused to GFP was used to couple the oleosome to an antibody for targeted delivery to breast cancer cells. The lipid core of the oleosome was loaded with magnetic nanoparticles and carmustine as the lipophilic drug. The magnetic oleosome was characterized using transmission electron microscopy and dynamic light scattering. Moreover, the specific delivery of oleosome into the target cancer cell was investigated via confocal microscopy. To examine the cell viability of the delivered oleosome, a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out. Furthermore, an animal study was conducted to confirm the effect resulting from the delivery of the anticancer drug-loaded oleosomes. Taken together, the fabricated lipophilic drug-loaded magnetic oleosome can be a powerful tool for oil-based drug delivery agent for cancer therapy.

Ultrasensitive detection of antibodies using a new Tus–Ter-lock immunoPCR system

A system consisting of a protein LG coated surface for the capture of mammalian antibodies (target), and an antigen fused to Tus and stoichiometrically linked to a DNA template via the Tus-Ter-lock sequence allowed the ultrasensitive detection of 5.5 attomol of target by real-time immunoPCR in complex media.

Binding capability of the enediyne-associated apoprotein to human tumors and constitution of a ligand oligopeptide-integrated protein

The molecule of lidamycin that belongs to the chromoprotein family of antitumor antibiotics is composed of an apoprotein (LDP) and an enediyne chromophore. The enediyne moiety of the molecule is responsible for the potent cytotoxicity; however, the biological function of the apoprotein moiety, particularly its interaction with cancer cells, remains unclear. In present study, the binding capability of LDP to human tumors was detected for the first time by tissue microarray. LDP bound to various human tumors with significant difference from the corresponding normal tissues. Positive correlation between binding activity and the overexpression of VEGF and EGFR was confirmed by lung carcinoma tissue microarray. A fusion protein LG–LDP that consists of LDP and a ligand oligopeptide to EGFR was constructed by DNA recombination. LG–LDP showed augmented binding to EGFR-overexpressing cancer cells. Furthermore, an energized fusion protein LG–LDP–AE was prepared by integrating the active enediyne (AE) into LG–LDP molecule. By MTT assay, LG–LDP–AE displayed extremely potent cytotoxicity to cancer cells with IC50 approximate to 0.01 nM. The results indicate that LDP binds to various human tumors and it might serve as a delivery carrier by integration of ligand oligopeptide to manufacture motif-based, targeted fusion proteins for cancer.

Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli

Different chimeric proteins combining the non-catalytic C-terminal putative cellulose binding domain of Clostridium cellulovorans endoglucanase-xylanase D (EngD) with its proline-threonine rich region PT-linker, PTCBD EngD, cellulose binding domain of C. cellulovorans cellulose binding protein A, CBD CbpA, cohesin domains Cip7, Coh6 and CipC1 from different clostridial species and recombinant antibody binding protein LG were constructed, expressed, purified and analyzed. The solubilities of chimeric proteins containing highly soluble domains Cip7, CipC1 and LG were not affected by fusion with PTCBD EngD. Insoluble domain Coh6 was solubilized when fused with PTCBD EngD. In contrast, fusion with CBD CbpA resulted in only a slight increase in solubility of Coh6 and even decreased solubility of CipC1 greatly. PTCBD EngD and Cip7-PTCBD EngD were shown to bind regenerated commercial amorphous cellulose Cuprophan. The purity of Cip7-PTCBD EngD eluted from Cuprophan was comparable to that purified by conventional ion exchange chromatography. The results demonstrated that PTCBD EngD can serve as a bi-functional fusion tag for solubilization of fusion partners and as a domain for the immobilization, enrichment and purification of molecules or cells on regenerated amorphous cellulose.

Chimeric protein for selective cell attachment onto cellulosic substrates

We have developed a fusion protein (CBD-LG) incorporating a cellulose-binding domain and an antibody binding domain, protein LG, to provide an adaptor molecule for cell separation with regenerated cellulose hollow fiber arrays. A single hollow fiber cell adhesion assay utilizing a CD34+ cell line, KG1a, was used to investigate whether ligand affinity interactions were strong enough for cell attachment and separation. CBD-LG efficiently captured CD34+ cells labeled with the mouse IgG2a monoclonal antibody MHCD3400. However, it was not possible to bind CD34+ cells labeled with an IgG1 antibody (HPCA-2). The low affinity of HPCA-2 for LG was overcome by secondary antibodies: KG1a cells that were dual labeled with HPCA-2 followed by rat anti-mouse IgG1 adhered inside hollow fibers coated with CBD-LG. Alternatively, immobilized rabbit polyclonal anti-mouse IgG1 captured cells labeled with HPCA-2. The development of an adaptor molecule to display recombinant domains at the surface of hollow fibers will be an effective tool to investigate cellular ligand-receptor interactions, a necessary step in the development of hollow fiber bioreactors for manufacture of human cellular products.

Engineered proteins containing the cohesin and dockerin domains from Clostridium thermocellum provides a reversible, high affinity interaction for biotechnology applications

The cohesin–dockerin interaction, which is responsible for the formation of the cellulosome complex of cellulolytic bacteria, is a calcium-dependent, high affinity interaction. In this study, the cohesin (Cip7) and dockerin (Doc) domains of Clostridium thermocellum were fused to the cellulose-binding domain (CBD) of C. cellulovorans and the antibody-binding domain, protein LG, respectively, to form CBD-Cip7 and LG-Doc. Immobilised CBD-Cip7 was able to bind LG-Doc and subsequently antibody as determined using surface plasmon resonance. Binding was reversed by the removal of Ca 2+ with EDTA. The dockerin containing fusion protein was affinity purified using an immobilised cohesin domain. Elution of the LG-Doc from the cohesin column was with EDTA. This affinity chromatography was repeated using an LG-dockerin column for the purification of cohesin fusion protein. The fusion proteins created in this report have shown that the properties of the cohesin and dockerin domains can be transferred to other protein domains and that the interaction between the cohesin and dockerin is specific, Ca 2+-dependent and reversible. We have shown that the cohesin–dockerin interaction has several properties making it suitable for use in recombinant fusion protein production and purification.

Temperature‐triggered purification of antibodies

In this article the unique capability of elastin-like protein (ELP) to reversibly precipitate was combined with the high affinity and specificity of antibody-binding domains such as Protein G, Protein L, or Protein LG as a general method for antibody purification that combines in a unique manner the simplicity and robustness of temperature-triggered precipitation with the selectivity of affinity interactions. In a single precipitation step, antibodies derived from different sources (animal sera or hybridoma cell cultures) were selectively recovered by a simple temperature trigger. Due to the versatility of the binding ligands toward different classes of antibodies, we believe that this technology will be useful as an economical, highly efficient, and universal platform for the purification of antibodies.

Characterization of the binding properties of protein LG, an immunoglobulin-binding hybrid protein.

Protein LG is a 50-kDa hybrid molecule containing four Ig-light-chain-binding domains from protein L of Peptostreptococcus magnus and two IgG-Fe-binding repeats from streptococcal protein G. Here we analyse the binding of protein LG to Ig from several mammalian species. Protein LG was shown to bind human IgG of all subclasses and other Ig classes that carry kappa chains. The binding to human IgG was only marginally influenced by changes in temperature (4-37 degrees C) or salt concentration (0-1.6 M), and was stable over a wide pH range (pH 4-10). Protein LG bound to Ig from 11 of 12 mammalian species, including those of rabbit, mouse and rat. The affinity constants obtained for the interactions between protein LG and polyclonal IgG from rabbit (4.0 x 10(9) M-1), mouse (1.7 x 10(9) M-1) and rat (1.3 x 10(9) M-1) were similar to the value previously reported for the interaction between the hybrid protein and human polyclonal IgG (5.9 x 10(9) M-1). The interaction between protein LG and a mouse IgG mAb was not influenced by the presence of the specific protein antigen, nor was the binding of this antibody to its ligand affected by protein LG. Inhibition experiments demonstrated that the Ig-binding site of one of the fusion partners retained its ligand-binding capacity when the other component was occupied. Protein LG selectively absorbed 85-90% of the total Ig present in human and rabbit sera and 75-80% of the Ig in sera from mouse and rat. Human serum depleted of C1q, factor D and properdin and preabsorbed by protein LG could be used as a source for other complement factors. These data demonstrate that protein LG is a very versatile Ig-binding protein.

Multiple Ligand Interactions for Bacterial Immunoglobulin‐Binding Proteins on Human and Murine Cells of the Hematopoetic Lineage

A group of bacterial Ig-binding surface proteins were studied: protein H and M1 are from Streptococcus pyogenes and interact with IgG, protein L is expressed by Peptostreptococcus magnus and shows affinity for Ig light chains, whereas protein LG is a chimeric construction combining the binding properties of protein L with the IgG-binding activity of protein G from group C and G streptococci. Proteins L and H coupled to Sepharose were mitogenic for human peripheral blood lymphocytes (PBL) and mouse splenic B cells, but not when added in soluble form. Differentiation to Ig secretion was induced by protein H-Sepharose in mouse splenic B cells but not in human PBLs. In FACS analysis FITC-labelled protein H stained virtually all CD19+ cells in human peripheral blood as well as a majority of the CD3+ population. Protein L bound the majority of the CD19+ population, but also a fraction of the CD19-/CD3 population. Protein M1 was not mitogenic but stained the entire CD19+ population and 70% of the CD3+ population. Identical staining patterns were observed with mouse splenocytes using B220 and T-cells receptor as lineage markers. The chimeric protein LG was a potent mitogen for mouse splenic B cells when added either coupled to Sepharose or in soluble form. In addition, protein LG induced differentiation to Ig secretion of the responding mouse splenic B cells. In FACS analysis, protein LG stained the entire CD19+ and the majority of the CD19-/CD3 lymphocyte population as well as all B220+ mouse splenocytes and a fraction of the splenic T cells. These data indicate that the bacterial proteins studied interact with surface structures of several leucocyte populations and can hence interfere with the immune system at multiple levels.

Recombinant proteins L and LG: efficient tools for purification of murine immunoglobulin G fragments.

In order to improve antibody purification methods, recombinant proteins L and LG were tested in the purification of murine monoclonal immunoglobulin G (IgG) and its fragments. After affinity constant evaluation in different buffer systems, high-performance affinity chromatographic columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of different isotypes. Affinity chromatographic experiments confirmed radioimmunoassay results showing that protein L bound 75% of the tested antibody fragments whereas protein LG had affinity for all the tested fragments. These results demonstrate that protein LG is the most powerful Ig-binding tool so far described.

Recombinant proteins L and LG. Two new tools for purification of murine antibody fragments.

Several bacterial cell wall proteins, like proteins A and G, with valuable affinity for immunoglobulins have been discovered and are currently employed in immunological techniques. In 1988, protein L, a bacterial cell wall protein with Ig-binding capacity, was isolated from the anaerobic bacterial species Peptostreptococcus magnus. Binding data with immunoglobulin fragments suggested that protein L could selectively bind the variable region of human kappa light chains. More recently a recombinant LG fusion protein was expressed in E. coli containing four repeated Ig-binding domains of protein L (fragment B1-4) and two IgG Fc-binding protein G domains (fragment CDC). Recombinant L and LG proteins were tested in the purification of murine monoclonal IgG and their fragments. After affinity-constant evaluation in different buffer systems, high-pressure affinity-chromatography columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of various isotypes. Affinity-chromatography experiments confirming radioimmunoassay results showed 75% fragment-binding capacity of protein L and 100% of protein LG. These results evidenced protein LG as the most powerful Ig-binding tool so far described. Therefore, application of these proteins may be suggested in the purification of murine immunoglobulins and their fragments, including the engineered ones.

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.