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S M Deyev

Publications and source records attributed to S M Deyev.

17 recordsLinked to original sources
PubMed2003

Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium Meiothermus ruber and characterization of the recombinant enzyme.

A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg2+ ions was required. Zn2+ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62 degrees C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60 degrees C, whereas at 80 degrees C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.

Alkaline Phosphatase↗
PubMed2000

The quantitative characteristics of efficiency of ballistic transfection of chimeric antibody genes.

A quantitative approach was applied to the study of in vivo expression of foreign genes introduced into mice by ballistic transfection. Because in some cases one must take into account both the level of synthesized protein and that of antibodies to it, we derived the equation which allows to calculate the exact quantity of both proteins. This formula was applied to in vivo expression of a chimeric (human/mice) immunoglobulin E gene. The immunochemical analysis using this equation showed that the Ig concentration succeeded 4, 6, 12 IU/ml and undetectable level, respectively, upon transfection in mouse liver, spleen, foot pad and ear cartilage.

Animals↗
PubMed2000

Antiferritin single-chain Fv fragment is a functional protein with properties of a partially structured state: comparison with the completely folded V(L) domain.

Differential scanning calorimetry and spectroscopic probes were applied to study folding and stability of the single-chain Fv fragment (scFv) of the anti-human ferritin antibody F11 and its isolated variable light-chain (V(L)) domain. The scFv fragment followed variable heavy-chain domain (V(H))-linker-V(L) orientation and contained (Gly(4)Ser)(3) linker peptide. The two proteins were produced in Escherichia coli and refolded from denaturant-solubilized inclusion bodies. The isolated V(L) domain demonstrated a typical immunoglobulin fold with well-defined secondary and tertiary structure and was capable of binding human ferritin with K(a) = 1.8 x 10(7) M(-)(1), approximately (1)/(30) of the affinity of the parent F11 antibody. Involvement of this V(L) domain into the two-domain scFv fragment yielded a distorted secondary and significantly destabilized tertiary structure in which neither of the two constituent domains attained complete folding. The thermal unfolding enthalpy of scFv F11 at pH 7.0 was as low as 5. 0 J.g(-)(1) versus 16.3 J.g(-)(1) obtained for the V(L) domain and 24.7 J.g(-)(1) for the parent F11 antibody (mouse IgG2a subclass). Intrinsic fluorescence and near-ultraviolet circular dichroic (CD) spectra, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonate, confirmed partial loss of tertiary interactions in scFv. The spectroscopic and calorimetric properties of scFv F11 under physiological conditions are consistent with a model of a partially structured state with a distorted beta-sheet as a secondary structure and partial loss of tertiary interactions, which closely resembles the alternatively folded A-state adopted by an immunoglobulin at pH 2-3 [Buchner, J., Renner, M., Lilie, H., Hinz, H.-J., Jaenicke, R., Kiefhaber, T., and Rudolph, R. (1991) Biochemistry 30, 6922-6929]. However, scFv F11 demonstrated only an approximately 4-fold decrease in the antigen-binding affinity (K(a) = 1.3 x 10(8) M(-)(1)) versus the parent F11 antibody. The scFv fragment F11 provides the first description of a functional protein trapped under physiological conditions in a partially structured state. This state is either close to the native one in the antigen-binding affinity or, alternatively, initial weak binding of the antigenic epitope induces folding of scFv F11 into a more structured conformation that generates relatively high affinity.

Amino Acid Sequence↗
PubMed1998

Antiferritin single-chain antibody: a functional protein with incomplete folding?

The pET(scF11) plasmid was constructed comprising the gene of a single-chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable V(H) domain, (Gly4Ser)3 linker peptide, and light chain variable V(L) domain. The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography. ScF11 demonstrated only a approximately 8-fold decrease in the affinity (Ka = 5.1 x 10(8) M(-1) in RIA and 1.8 x 10(8) M(-1) in ELISA) vs. the parent IgG2a/kappa monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant-induced fluorescence 'dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS) recognizing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single-chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding.

Amino Acid Sequence↗
PubMed1998

Ribonuclease-charged vector for facile direct cloning with positive selection.

Plasmid vectors for positive selection of cloned inserts in Escherichia coli were devised, based on an expression plasmid (pMT416) for the bacterial ribonuclease barnase. In addition to the barnase gene under control of a synthetic tac promoter, these plasmids carry the gene for the barnase inhibitor, barstar, the constitutive expression of which protects the bacterium from the detrimental effects of moderate barnase production. Full expression of the barnase gene overcomes protection by barstar and becomes lethal. Having a unique SmaI/XmaI site in the barnase structural gene, pMT416 itself can be used as a selective vector: uncut or religated pMT416 will preclude growth while plasmids with inserts in the barnase gene will allow the cells to survive. The entire pUC polylinker was inserted into the barnase gene in place of the Val-36 codon. This insert of nineteen largely hydrophilic amino acids does not prevent the lethal effect of full expression of the gene. The resulting plasmid, pMT440, is a generally useful selective cloning vector representing the "kill-the-rest" approach.

Amino Acid Sequence↗
PubMed1996

Recombinant barnase as a label in ELISA.

Recombinant barnase was proposed as a label in the enzyme-linked immunosorbent assay (ELISA). Barnase-conjugated pig transferrin was prepared by the periodate oxidation procedure. Solid-phase bound barnase activity was determined from the change in RNA-ethidium bromide complex fluorescence upon RNA hydrolysis. The sensitivity of transferrin-barnase conjugate determination in ELISA was no less than 5 ng per well. The conjugate was applied in competition ELISA for free transferrin determination.

Animals↗
PubMed1996

A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene.

A plasmid vector with a multiple cloning site (MCS) for positive selection of cloned inserts in Escherichia coli (Ec) has been devised, based on the expression plasmid (pMT416) for the bacterial ribonuclease barnase (Barn). The host is protected from the lethal effect of moderate expression of barn by expression of the gene bars, encoding its inhibitor, barstar (Bars), placed on the same plasmid. Full expression, however, is lethal. Induction is also lethal with the derived plasmid, pMT440, which has the pUC19 MCS inserted into barn. Under inducing conditions, transformation by the vector is lethal unless the product of the modified barn is inactivated by insertion of cloned DNA fragments into the MCS. Plasmid pMT440 is, therefore, a generally useful selective cloning vector not requiring any special strain of Ec.

Amino Acid Sequence↗
PubMed1994

Group-selective immunoassay.

A new solid-phase immunoassay technique has been applied for anti-digoxin monoclonal antibodies (Mabs) detection. At the first assay step, anti-digoxin Mabs (IgG1, Kaff = 9.2 x 10(9) M-1) were bound to a specially prepared immunosorbent, the microtiter plates coated with digoxin-human serum albumin conjugate (Dig-HSA), in which free amino groups were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. After antigen-antibody reaction, free amino groups were located only on the anti-digoxin Mabs, bound to chemically modified immunosorbent. At the second assay step, free amino groups of anti-digoxin Mabs were biotinylated by N-hydroxysuccinimide-biotin ester. Then the biotin residues were detected by the streptavidin-peroxidase conjugate. The method does not require second labeled antibodies and may be used for anti-hapten hybridoma screening.

Antibodies, Monoclonal↗
PubMed1993

Production of recombinant antibodies in lymphoid and non-lymphoid cells.

A recombinant tandem of 'chimeric' mouse/human immunoglobulin (Ig) genes was constructed and inserted into plasmid pGEM1 under the control of the T7 bacteriophage RNA polymerase promoter. Lymphoid (Sp2/0) and non-lymphoid (CHO) cell lines used for transfection contained in their genomes a semisynthetic gene of T7 RNA polymerase and steadily expressed this enzyme. It was shown for the first time that a stable polycystronic transcription of the Ig gene tandem occurs under the control of a single T7 phage promoter, both in lymphoid and non-lymphoid cells. Synthesis of kappa-light and epsilon-heavy Ig chains and functionally active antibodies was observed in the above-mentioned transfected cell lines.

Animals↗
PubMed1991

Allelic variants of rearranged immunoglobulin heavy and light chain genes in hybridoma PTF-02 and parent myeloma.

The hybridoma PTF-02 secretes an antibody against pig transferrin. Rearranged genes for heavy and light immunoglobulin chains have been studied in the genomes of this hybridoma and in the parent myeloma P3-X63.Ag8.653. The hybridoma was shown to contain three rearranged allelic variants of the heavy chain gene's locus. The gene H2, responsible for synthesis of the heavy chain of the antibody to transferrin, was transmitted in the hybridoma cell from a lymphocyte. Two other genes (H1 and H3) were found both in the hybridoma and parent myeloma genomes. The gene H1 was identified in MOPC21 myeloma, which is a precursor of the X63.Ag8 descendent line. Rearranged k genes were also identified both in the hybridoma and parent myeloma. A functional (K2) gene and a fetal (F) gene appeared in the hybridoma genome from an antigen-stimulated normal lymphocyte. The fetal gene was lost in the course of continuous cultivation of the hybridoma PTF-02 cell line. The gene K1 was transmitted from the myeloma used for fusion. In such a way, the pedigree of rearranged heavy and light chain genes in the hybridoma PTF-02 was established. The results obtained in this work may be relevant to many hybridomas whose immortalizing fusion partner is a MOPC21 derivative, and allow one to identify and isolate functional variable genes to create recombinant constructions.

Alleles↗
PubMed1986

Immunoglobulin K chain genes in mouse hybridoma PTF-02 and parent myeloma. Insertion of hybridoma K gene into the plasmid pSV2-gpt.

The mouse myeloma line P3-X63-Ag8.653 currently used as the parent line for hybridoma construction contains only one (non-productive) gene for immunoglobulin K chains. The allelic gene is lost. In the mouse hybridoma PTF-02 two K genes can be found. One is identical with the gene of the myeloma parent line, the other originates in lymphocytes and is transcribed and translated in the K chain of the antibody secreted by the hybridoma. From the gene library of hybridoma PTF-02 in phage Charon 28 both K genes were isolated. Restriction endonuclease mapping and Southern blot hybridization demonstrated that the fragment comprising the lymphocyte gene was of the size (7.5 kb) sufficient to carry all exons, transcription and translation units. This gene was then recloned in the plasmid pBR322 and shuttle plasmid pSV2-gpt, which opens up possibilities for transfection of lymphoid cells and for study of the regulation of individual gene expression.

Animals↗
PubMed1984

Purification of mRNA for immunoglobulin kappa-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA.

The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin kappa-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3'-untranslated region and a part of the constant region of the kappa-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52 degrees C and by elution at 60 degrees C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the kappa-chain, was identified as the only translation product.

Animals↗
PubMed1978

Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene. Cross-link localization.

At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the epsilon-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 A (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5'-phosphate in the active site.

Animals↗
PubMedDate not supplied

Expression of immunoglobulin genes tandem in eukaryotic cells under the control of T7 bacteriophage RNA polymerase.

A tandem of recombinant mouse/human immunoglobulin (Ig) genes was constructed and inserted into the plasmid pGEM1 under the control of T7 phage RNA polymerase promoter. Sp2/0 lymphoid cell line and Chinese Hamster Ovary (CHO) cells were used as the targets for gene transfection. Both cell lines contained in their genomes a T7 RNA polymerase gene modified with a nuclear-located signal derived from SV40 large T-antigen. Cell lines transfected with the gene tandem effectively synthesized mRNA (up to 9 x 10(3) bp) that hybridized with epsilon- and kappa-gene probes. Separate transcripts corresponding to mRNAs of individual heavy and light chains were not detected in either transfected cell line. It follows from these data that transcription in the transfected cells is controlled mainly by the T7 phage polymerase promoter. Both lymphoid and nonlymphoid cell lines transfected with the gene tandem synthesized the epsilon-heavy (70 kDa) and kappa-light (25 kDa) Ig polypeptide chains. Production of chimeric antibodies by the myeloma Sp2/0 cells was higher than that by the CHO cells. Individual clones synthesized up to 150 ng/mL chimeric IgE. However, only lymphoid Sp2/0 cells were capable of efficient secretion of the recombinant antibodies. The mechanism of translation of mRNA synthesized in eukaryotic cells by T7 phage RNA polymerase is discussed.

Amino Acid Sequence↗