Non-conjugated Protein Research Articles

Elution Profiles of Antibody-Drug Conjugates in Preparative Chromatography

Monoclonal antibody drug conjugate (ADCs) have received much attention as pharmaceutical agents for treating serious diseases such as cancer. However, it is difficult to separate them on the basis of the drug to antibody ratio, DAR. Hydrophobic chromatography (HIC) is commonly used for the analysis of the drug to antibody ratio, DAR. The retention of ADCs on HIC can be controlled by the hydrophobic nature of ADCs, depending on the mobile phase conditions. They are sometimes performed at the restricted conditions where the solubility is too low. Ion exchange chromatography (IEC) using electrostatic interaction is an orthogonal method to HIC. IEC is widely used because of its higher capacity than HIC. We investigated the retention behavior of the protein conjugated with surrogate drugs on IEC. The surrogate drugs employed are 7-diethylamino-3-(4’-maleimidylhenyl) 4-methylcoumarin (CPM), N-(1-pyrenyl) maleimide (NPM). Bovine serum albumin (BSA) was used as a model protein. The molar ratio (CPM and NPM to protein) was set to 3. The maleimide group of CPM and NPM reacts with the thiol group of the proteins. On the linear gradient elution experiments, the elution salt concentrations of the conjugated and non-conjugated proteins were measured to obtain chromatographic parameter of the number of binding sites, B.

Stability and refolding of Dihydrofolate reductase enhances with nano-conjugation

The nano-conjugation of proteins is an active area of research due to potential biomedical and nanotechnological applications. Many protein-nanoconjugates were designed for various applications, such as drug delivery, molecular imaging, and liquid biopsy etc. However, the challenges remain to ensure protein stability and to retain the conformational state of the protein intact upon nano-conjugation. In this communication we have reported the status of stability and refolding ability of Au-NP conjugated zDHFR protein. The effect of nano-conjugation of zDHFR on the thermal stability and it's refolding from thermally denatured state have been extensively studied. Zebrafish Dihydrofolate reductase (zDHFR) is an essential enzyme which acts as a crucial part in synthesis of purine, thymidylate and various amino acids in cells. We have nano-conjugated zDHFR protein with Au-nanoparticles and studies were conducted for thermally denatured Au-NP conjugated zDHFR and compared with the non-conjugated protein. Refolding experiment of heat denatured Au-NP conjugated zDHFR was carried out to check the status of refolding and the result was compared with the non-conjugated protein. Our observation reveals that nano-conjugation stabilises the zDHFR protein against thermal denaturation. Furthermore, the nano-conjugation promotes refolding process of thermally unfolded DHFR such that the yield of refolding substantially increases.

A novel pH sensor with application to milk based on electrochemical oxidative quinone-functionalization of tryptophan residues

The electrochemical oxidative quinone-functionalization of tryptophan (Trp) residues, and the proton-coupled electron transfer properties between quinonized Trp (Trp=O) and its re-reduced species (Trp-OH) are investigated for potentiometric pH sensors. The Trp=O/Trp-OH couples are assembled on a graphite electrode using multiple cyclic voltammetry, and exhibit a pair of well-defined redox peaks at redox potential of 0.090 V (vs. SCE) at pH 7.0 and 298 K. The proton-coupled electron transfer properties of Trp=O/Trp-OH couples are dependent on electrode substrate material, incubation temperature and pH values, and Trp residue sources including amino acid, peptide and protein. The Trp=O/Trp-OH-based surface-confined exothermic electrode reaction rates show a decrease with increasing incubation temperature from 283 to 323 K at pH 5.0. Both of redox peak potential and open-circuit potential under optimum conditions have a good linear pH dependence between 1.0 and 12.0 with sensitivity of more than 52 mV pH −1 and relative standard derivation of 1.5%. The proposed potentiometric pH sensor has significant advantages of low cost, good stability and reproducibility, and strong anti-interference ability. The present study shows a new approach to endow Trp, Trp-containing peptides and non-conjugated proteins with analogous proton-coupled electron transfer performance to prosthetic groups of quinoproteins for monitoring pH changes in milk samples.

Determination of conjugated protein on nanoparticles by an adaptation of the Coomassie blue dye method

A new accurate spectrophotometric method for protein determination on nanoparticles is described. The method is based on the Coomassie blue dye that binds to the basic and aromatic amino acid residues of proteins, especially arginine and lysine. A known amount of reagent dye was mixed with a variety of protein-loaded nanoparticles. Thereafter the unconjugated reagent was mixed with excess protein (bovine serum albumin) and titrated. In this method, the reacted dye on the protein coating of nanoparticle is directly determined, in opposite to the conventional method, in which the conjugated protein is determined as the difference between the non-conjugated protein found in the supernatant after centrifugation, and the total amount of protein originally used. This method is able to measure amounts of coated protein lower than 1 ppm.•Simple and accurate method especially adapted for protein-coated nanoparticles.•The amino acid residues of protein in the nanoparticle surface react with Coomassie brilliant blue dye.•The unreacted dye is titrated with an excess of a standard protein.

Multi-walled carbon nanotubes functionalized with recombinant Dengue virus 3 envelope proteins induce significant and specific immune responses in mice

BackgroundDengue is the most prevalent arthropod-borne viral disease in the world. In this article we present results on the development, characterization and immunogenic evaluation of an alternative vaccine candidate against Dengue.MethodsThe MWNT-DENV3E nanoconjugate was developed by covalent functionalization of carboxylated multi-walled carbon nanotubes (MWNT) with recombinant dengue envelope (DENV3E) proteins. The recombinant antigens were bound to the MWNT using a diimide-activated amidation process and the immunogen was characterized by TEM, AFM and Raman Spectroscopy. Furthermore, the immunogenicity of this vaccine candidate was evaluated in a murine model.ResultsImmunization with MWNT-DENV3E induced comparable IgG responses in relation to the immunization with non-conjugated proteins; however, the inoculation of the nanoconjugate into mice generated higher titers of neutralizing antibodies. Cell-mediated responses were also evaluated, and higher dengue-specific splenocyte proliferation was observed in cell cultures derived from mice immunized with MWNT-DENV3E when compared to animals immunized with the non-conjugated DENV3E.ConclusionsDespite the recent licensure of the CYD-TDV dengue vaccine in some countries, results from the vaccine’s phase III trial have cast doubts about its overall efficacy and global applicability. While questions about the effectiveness of the CYD-TDV vaccine still lingers, it is wise to keep at hand an array of vaccine candidates, including alternative non-classical approaches like the one presented here.

Emulsifying Properties of Food Proteins Conjugated with Glucose or Lactose by Two Methods (Spray-Drying Or Freeze-Drying)

Emulsifying properties of two commercial food proteins (plasma protein or egg albumen) conjugated with two sugars (glucose or lactose) by spray-drying or lyophylization were studied. Changes in protein structure were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sulfhydryl group, Fourier transform infrared spectroscopy and differential scanning calorimetry, emulsion properties (emulsion capacity and stability, emulsion work, and emulsifying activity index). Protein conjugation increased proteins molecular weight and decreased sulfhydryl groups, coinciding with lower denaturation enthalpies, corroborating structural changes by conjugation with glucose or lactose by either spray-drying or freeze-drying. Glucose conjugation of the proteins employed at the experimental conditions resulted in structural changes that emulsify more oil with no detrimental effect on emulsion superficial area formation. Although freeze-drying conjugation improved emulsifying properties, spray-drying also enhanced these properties, as compared to non-conjugated proteins. Spray-drying was a faster way to conjugate food proteins via Maillard reaction with improved emulsifying properties.

Electrochemistry of nonconjugated proteins and glycoproteins. Toward sensors for biomedicine and glycomics.

In this review, we wish to show that in the recent years a significant progress was done in the EC analysis of practically all proteins, based on electroactivity of amino acid (aa) residues in proteins. Also electrochemistry of polysaccharides, oligosaccharides and glycoproteins greatly advanced in creating important steps for its larger application in the glycoprotein research. In recent decades, a great effort was devoted to the discovery and application of biomarkers for analysis of different diseases, including cancer. In the following paragraphs, special attention will be paid (i) to intrinsic electroactivity of peptides and proteins, including the sensitivity to changes in protein 3D structures, as well as to recent advances in EC investigations of DNA-protein interactions, (ii) to intrinsic electroactivity of glycans and polysaccharides, advances in EC detection of lectin-glycoprotein interactions and to introduction of electroactive labels to polysaccharides and glycans and finally (iii) to EC detection of protein biomarkers, based predominantly on application of antibodies in immunoassays, nucleic acid and peptide aptamers for construction of aptasensors, and lectin biosensors for detection of glycoprotein biomarkers.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

Electrochemistry of Nucleic Acids and Non-conjugated Proteins in Biomedicine

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Auto-phosphorylation of a voltage-gated K+ channel controls non-associative learning

Here, we characterize a new K(+) channel-kinase complex that operates in the metazoan Caenorhabditis elegans to control learning behaviour. This channel is composed of a pore-forming subunit, dubbed KHT-1 (73% homology to human Kv3.1), and the accessory subunit MPS-1, which shows kinase activity. Genetic, biochemical and electrophysiological evidence show that KHT-1 and MPS-1 form a complex in vitro and in native mechanosensory PLM neurons, and that KHT-1 is a substrate for the kinase activity of MPS-1. Behavioural analysis further shows that the kinase activity of MPS-1 is specifically required for habituation to repetitive mechanical stimulation. Thus, worms bearing an inactive MPS-1 variant (D178N) respond normally to touch on the body but do not habituate to repetitive mechanical stimulation such as tapping on the side of the Petri dish. Hence, the phosphorylation status of KHT-1-MPS-1 seems to be linked to distinct behavioural responses. In the non-phosphorylated state the channel is necessary for the normal function of the touch neurons. In the auto-phosphorylated state the channel acts to induce neuronal adaptation to mechanical stimulation. Taken together, these data establish a new mechanism of dynamic regulation of electrical signalling in the nervous system.

Electroactivity of Nonconjugated Proteins and Peptides. Towards Electroanalysis of All Proteins

AbstractPresent proteomics and biomedicine require sensitive analytical methods for all proteins. Recent progress in electrochemical analysis of peptides and proteins based on their intrinsic electroactivity is reviewed. Tyrosine and/or tryptophan‐containing proteins are oxidizable at carbon electrodes. At mercury electrodes all peptides and proteins (about 13 peptides and >25 proteins were tested) produce chronopotentiometric peak H at nanomolar concentrations. This peak is sensitive to changes in protein structure. Microliter sample volumes are sufficient for the analysis. Electrochemical methods can be used in studies of nucleic acid‐protein interactions and can be applied in biomedicine. Examples of such applications in neurogenerative diseases and cancer are presented.

Preparation and characterization of interleukin-2-gelonin conjugates made using different cross-linking reagents.

Conjugates of IL-2 with the ribosome-inactivating protein gelonin were prepared using heterobifunctional reagents to link the proteins via disulfide, acid-labile, and noncleavable linkers. In each case, one protein was modified using 2-iminothiolane. The sulfhydryl groups so introduced were then reacted either with 2-nitro-5-dithiobenzoate groups or with iodoacetamido groups which had been introduced into the second protein. In the case of the acid-labile linkage, a reagent which forms a labile bond upon reaction with amino groups, 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride (its synthesis is described in this paper) was used to modify the toxin. The conjugates were separated from nonconjugated proteins by gel filtration on Sephadex G100 (SF). Each was analyzed with respect to its ribosome-inactivating activity, its ability to bind to the IL-2 receptor, and its in vitro cytotoxicity. The ribosome-inactivating activity of gelonin was unaffected by modification with 2-iminothiolane and was retained in conjugates prepared using this reagent. Modification of the toxin with 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride to form the acid-labile link drastically reduced the activity of the toxin. However, the activity of the toxin was recovered following acid treatment to release the native protein. Conjugates containing each type of linkage exhibited both specific binding and selective cytotoxicity toward cells expressing the IL-2 receptor. The most potent of these toxins, that containing the disulfide linkage, exhibited a cytotoxicity which was 2 orders of magnitude greater than that of unconjugated gelonin.

Determination of the partial specific volume of conjugated proteins

Specific volume quantities of macromolecules are of importance in connection with a variety of physico-chemical and biochemical techniques, such as analytical ultracentrifugation and small-angle scattering, e.g., for the determination of molecular weights in two- and multicomponent solutions. Specific volumes may be obtained from density experiments, calculations, or reliable approximations. Experimental values for the partial specific volume (¯v) of native conjugated proteins in aqueous solution vary in a wide range (0.59-1.05 cm3/g), while the values for nonconjugated proteins cluster around 0.735 cm3/g, as follows from a thorough investigation of the relevant literature. Volumes of conjugated proteins are substantially influenced by the nature and content of the nonprotein moiety. Typical glyco-, nucleo-, and metalloproteins show ¯v- values lower than those found for nonconjugated proteins, while lipoproteins exhibit high values. The ¯v-values of conjugated proteins in dilute salt or buffer solution (≤0.2 M) may be assumed to be composed additively from compositional increments. Based on the composition and assumed average values for the partial specific volumes of the main constituents of conjugated proteins (¯va=0.54, 0.61, 0.735, and 1.02 cm3/g for the nucleic acid, carbohydrate, protein, and lipid moieties, respectively), reliable calculations can be performed if some precautions are taken into account. In this context the theoretical background will be discussed, especially with respect to ultracentrifugal experiments in multicomponent solutions.