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Biomedical subjects

S A Narang

Publications and source records attributed to S A Narang.

At least 19 recordsLinked to original sources
PubMed1991

Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.

A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.

Amino Acid Sequence↗
PubMed1991

Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.

The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.

Amino Acid Sequence↗
PubMed1990

Synthesis and expression in Escherichia coli of DNA encoding the murine lambda 1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen.

A 658 bp DNA sequence corresponding to the murine lambda 1 chain of a monoclonal antibody, Se155-4, specific for the Salmonella serotype B O-antigen, was designed using Escherichia coli preferred codons and chemically synthesized by ligation of synthetic fragments into a linearized plasmid followed by transformation into E. coli. A synthetic signal peptide (ompA) was fused to express the L chain as a free polypeptide into the periplasm of E. coli cells. After isolation and purification, heterologous recombination of the E. coli L chain with mouse H chain gave an active antigen-binding protein. The activity was 15-20% when compared to protein created by an equivalent association of isolated natural mouse L and H chains as measured by a direct EIA assay. In inhibition experiments with the polysaccharide antigen, the two proteins showed identical titration curves and 50% inhibition points, indicating comparable KA values.

Amino Acid Sequence↗
PubMed1979

Synthesis of the human insulin gene. Part II. Further improvements in the modified phosphotriester method and the synthesis of seventeen deoxyribooligonucleotide fragments constituting human insulin chains B and mini-CDNA.

The purification of protected deoxyribooligonucleotides containing phosphotriester internucleotidic linkages has been improved by developing a deactivated silica gel chromatographic technique. The efficiency of this technique as applied in the modified phosphotriester approach has been demonstrated in the rapid synthesis of seventeen pure fragments constituting the sequence of human insulin B and mini-C DNA. The sequence of each oligomer was confirmed by the two-dimensional mobility shift method of fingerprinting.

Base Sequence↗
PubMed1979

Synthesis of human insulin gene. Part I. Development of reversed-phase chromatography in the modified triester method. Its application in the rapid and efficient synthesis of eight deoxyribooligonucleotides fragments constituting human insulin A DNA.

Preparative reversed-phase thin layer chromatography on silanized silica-gel (RP-2 and RP-18) has been developed to purify triester deoxyribooligonucleotides prepared by the modified triester method. The effectiveness of this technique has been demonstrated in the rapid synthesis of eight pure deoxyribooligonucleotides constituting the sequence of human insulin A DNA. The sequence of each of the deoxyribooligonucleotides was confirmed by the two-dimensional mobility-shift method of finger-printing.

Amino Acid Sequence↗
PubMed1977

Minimal length of the lactose operator sequence for the specific recognition by the lactose repressor.

A number of specific duplex DNA sequences which correspond to varying lengths of the lactose operator region have been synthesized by a combination of chemical and enzymatic methods. Repressor binding studies on these synthetic duplex operator molecules show that all the nucleotides essential for full lactose operator-repressor interactions are included in a 17-nucleotide-long duplex DNA that constitutes the minimal recognition sequence for this DNA-protein interaction.

Base Sequence↗
PubMed1977

Arylsulfonyltetrazoles, new coupling reagents and further improvements in the triester method for the synthesis of deoxyribooligonucleotides.

The modified triester approach has been further improved and refined to the synthesis of defined sequences of deoxyribo-oligonucleotides. Improvements include arylsulfonyltetrazoles as faster and milder condensing agents, benzenesulfonic acid to avoid depurination during deblocking of trityl protecting groups and improved chromatographic procedures for purification of triester intermediates and purification of the final product containing 3'-5' phosphodiester linkages.

DNA Restriction Enzymes↗
PubMed1977

A rapid and convenient synthesis of poly-thymidylic acid by the modified triester approach.

By using anhydrous triethylamine-pyridine to selectively remove the cyanoethyl group from the fully protected oligonucleotide, a substantial improvement has been achieved in yields and the rates of condensation by the modified triester approach from the 5' leads to 3' end. The unreacted oligonucleotide containing the 5'-hydroxy group was removed by treatment with bis (triazolyl)-p-chlorophenyl phosphate after each condensation in situ. These modifications, as exemplified by the synthesis of fully protected T12, T18, T24 and T38 in 80%, 77%, 70% and 50% yields respectively, should allow the ready synthesis of polynucleotides of even longer chain lengths by purely chemical methods.

Chemical Phenomena↗
PubMed1976

Cloned synthetic lac operator DNA is biologically active.

A chemically synthesised duplex DNA fragment containing the sequence of the lac operator was cloned in E. coli using the vehicle pMB9. Clones containing lac-pMB9 hybrid DNA produced beta-galactosidase constitutively and the hybrid DNA bound the lac repressor specifically.

Bacterial Proteins↗
PubMed1976

Chemical and enzymatic synthesis of lactose operator of Escherichia coli and its binding to lactose repressor.

The 21-nucleotide-long duplex DNA constituting the lactose operator sequence of E. coli has been synthesized by both chemical and enzymatic methods. The synthetic duplex has the essential feature of the lactose operator as seen by its binding to the lactose repressor. The binding of the synthetic operator fragment to the lactose repressor is specific because it is inhibited by the inducing ligand isopropyl-beta-D-thiogalactoside. Thus, it is now possible to show that a chemically synthesized oligodeoxynucleotide can be specifically recognized by its natural regulatory protein.

Bacterial Proteins↗
PubMed1976

Separation of potato spindle tuber viroid ribonucleic acid from Scopolia sinensis into three infectious forms and the purification and oligonucleotide pattern of fraction II RNA. Part IX.

The existence of three infectious forms of potato spindle tuber viroid (PSTV) RNA from Scopolia sinensis was demonstrated by fractionation with high salt, by reverse phase and high pressure liquid chromatography, and by polyacrylamide gel electrophoresis. Purification of fraction II was achieved by the following steps: extraction of nucleic acid with phenol, precipitation of the RNA with cetyltrimethylammonium bromide, fractionation of the RNA with lithium chloride and isopropanol, and finally gel electrophoresis. A procedure using reverse phase chromatography was developed to obtain 70-90% recovery of RNA from polyacrylamide gels. Purified PSTV fraction II RNA was digested with ribonuclease A and T and labelled with [gamma-32P[ATP using polynucleotide kinase. The labelled digests were separated by the electrophoresis-homochromatography procedures of Sanger. About 20 and 30 spots were obtained with ribonuclease A and T, respectively.

Chromatography, High Pressure Liquid↗