Production of Recombinant Human Transferrin in Eukaryotic Pichia pastoris Expression System.

The development and manufacturing of serum-free culture media allowing reducing the costs of preparations and standardizing the biotechnological process are important trends in biotechnology. Substitution of protein compounds in the serum-free media with recombinant analogues reduces the risk of contamination with various infectious agents. Human transferrin is a protein component of serum-free media responsible for the transport of Fe3+ ions into cells. We generated a producing strain P. pastoris secreting human transferrin to the culture medium. The use of constitutive GAP promoter and maintenance of medium pH at 6.5 allows attaining maximum level of transferrin expression (20 mg/liter).

Mouse Model for Assessing the Subchronic Toxicity of Organophosphate Pesticides.

The development of antidotes to organophosphate poisons is an important aspect of modern pharmacology. Recombinant acetylcholinesterase and butyrylcholinesterase are effective DNA-encoded acceptors of organophosphate poisons and, in particular, pesticides. Here, we present the results of a study on the effectiveness of recombinant butyrylcholinesterase (BChE) in modeling organophosphate poisoning caused by oral administration of paraoxon at a dose of 2 mg / kg. The study showed a high activity of BChE as a protective agent for subchronic anticholinesterase poisoning in an in vivo model. The administration of BChE in a dose of 20 mg / kg allows one to avoid mortality, and also contributed to rapid recovery after model poisoning.

[Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates].

Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^(-1)М^(-1) for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.

Application of Tetrameric Recombinant Human Butyrylcholinesterase as a Biopharmaceutical for Amelioration of Symptoms of Acute Organophosphate Poisoning.

We present a procedure for optimizing the expression of recombinant tetrameric butyrylcholinesterase that enables large-scale production with the yield >30 mg/liter (>90 mg/roller bottle). Intravenous injection of the preparation significantly increased survival and decreased the severity of symptoms of poisoning with paraoxon, an organophosphorus toxin.

Specific Depletion of Myelin-Reactive B Cells via BCR-Targeting.

B cells play a crucial role in the development and pathogenesis of systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce antibodies, but also secrete pro-inflammatory cytokines and present specific autoantigens to T cells. The treatment of autoimmune diseases via the elimination of the majority of B cells using the monoclonal anti-CD19/20 antibody (Rituximab) causes systemic side effects and, thus, requires a major revision. Therapeutic intervention directed towards selective elimination of pathogenic autoreactive B cells has the potential to become a universal approach to the treatment of various autoimmune abnormalities. Here, we developed a recombinant immunotoxin based on the immunodominant peptide of the myelin basic protein (MBP), fused to the antibody Fc domain. We showed that the obtained immunotoxin provides selective in vivo elimination of autoreactive B cells in mice with experimental autoimmune encephalomyelitis. The proposed conception may be further used for the development of new therapeutics for a targeted treatment of multiple sclerosis and other autoimmune disorders.

Chemical Polysialylation and In Vivo Tetramerization Improve Pharmacokinetic Characteristics of Recombinant Human Butyrylcholinesterase-Based Bioscavengers.

Organophosphate toxins (OPs) are the most toxic low-molecular compounds. The extremely potent toxicity of OPs is determined by their specificity toward the nerve system. Human butyrylcholinesterase (hBChE) is a natural bioscavenger against a broad spectrum of OPs, which makes it a promising candidate for the development of DNA-encoded bioscavengers. The high values of the protective index observed for recombinant hBChE (rhBChE) make it appropriate for therapy against OP poisoning, especially in the case of highly toxic warfare nerve agents. Nevertheless, large-scale application of biopharmaceuticals based on hBChE is restricted due to its high cost and extremely rapid elimination from the bloodstream. In the present study, we examine two approaches for long-acting rhBChE production: I) chemical polysialylation and II) in-vivo tetramerization. We demonstrate that both approaches significantly improve the pharmacokinetic characteristics of rhBChE (more than 5 and 10 times, respectively), which makes it possible to use rhBChE conjugated with polysialic acids (rhBChE-CAO) and tetrameric rhBChE (4rhBChE) in the treatment of OP poisonings.

Excessive Labeling Technique Provides a Highly Sensitive Fluorescent Probe for Real-time Monitoring of Biodegradation of Biopolymer Pharmaceuticals in vivo.

Recombinant proteins represent a large sector of the biopharma market. Determination of the main elimination pathways raises the opportunities to significantly increase their half-lives in vivo. However, evaluation of biodegradation of pharmaceutical biopolymers performed in the course of pre-clinical studies is frequently complicated. Noninvasive pharmacokinetic and biodistribution studies in living organism are possible using proteins conjugated with near-infrared dyes. In the present study we designed a highly efficient probe based on fluorescent dye self-quenching for monitoring of in vivo biodegradation of recombinant human butyrylcholinesterase. The maximum enhancement of integral fluorescence in response to degradation of an intravenously administered enzyme was observed 6 h after injection. Importantly, excessive butyrylcholinesterase labeling with fluorescent dye results in significant changes in the pharmacokinetic properties of the obtained conjugate. This fact must be taken into consideration during future pharmacokinetic studies using in vivo bioimaging.

Recombinant human butyrylcholinesterase as a new-age bioscavenger drug: development of the expression system.

Butyrylcholinesterase (BChE) is a serine hydrolase (EC 3.1.1.8) which can be found in most animal tissues. This enzyme has a broad spectrum of efficacy against organophosphorus compounds, which makes it a prime candidate for the role of stoichiometric bioscavenger. Development of a new-age DNA-encoded bioscavenger is a vival task. Several transgenic expression systems of human BChE were developed over the past 20 years; however, none of them has been shown to make economic sense or has been approved for administration to humans. In this study, a CHO-based expression system was redesigned, resulting in a significant increase in the production level of functional recombinant human butyrylcholinesterase as compared to the hitherto existing systems. The recombinant enzyme was characterized with Elman and ELISA methods.

[Binding of phosphatidylinositol-3,4,5-triphosphate to 45-kDa sec14-like protein from the rat olfactory epithelium in liposomes].

The binding of phosphatidylinositol-3,4,5-triphosphate to a protein with molecular mass of 45 kDa from rat olfactory epithelium (p45) was investigated using a model membrane system. Liposomes containing a mixture of phospholipids (phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol-3,4,5-triphosphate) were used in the study. The binding of the protein to liposomes caused by its interaction with phosphatidylinositol-3,4,5-triphosphate was confirmed by cosedimentation and immunoblotting with chemiluminescent detection using monoclonal antibodies to the native protein p45. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

Endogenous tumour necrosis factor-alpha sensitise melanoma cells to glucosaminylmuramyl dipeptide.

Flow cytometry was used to demonstrate that cultured human melanoma BRO cells expressed membrane-bound tumour necrosis factor-alpha (TNF-alpha) and were able to release TNF-alpha upon treatment with glucosaminylmuramyl dipeptide (GMDP). The released TNF-alpha was shown to prime melanoma cells, previously unable to respond to GMDP by increasing expression of melanoma-associated antigens, making them sensitive to GMDP treatment.

[Preparation and characteristics of monoclonal antibodies against GTP-binding protein from the bovine retina]. / Poluchenie i kharakteristika monoklonal'nykh antitel k GTP-sviazyvaiushchemu belku iz setchatki glaz byka.

Five monoclonal antibodies against the native GTP-binding protein (transducin) from bovine retina have been prepared. By immunoblotting and immunoenzymatic analysis of the isolated alpha- and gamma-subunits of transducin and the beta gamma-subunit complex it was determined that two monoclonal antibodies A3G7 and A3C10 recognize linear antigenic determinants on the alpha-subunit, two other, A3E4 and 3B3, bound specifically to the gamma-subunit, and monoclonal antibodies 1C3 interact only with native transducin. Both antibodies against the alpha-subunit inhibited transducin GTPase activity, whereas antibodies A3E4, 3B3 and 1C3 did not affect it.