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Biomedical subjects

G R Nakayama

Publications and source records attributed to G R Nakayama.

At least 19 recordsLinked to original sources
PubMed2001

Combinatorial chemistry. Web alert.

A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Chemical Biology.

Combinatorial Chemistry Techniques↗
PubMed2000

Web alert.

A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Chemical Biology.

Chemistry Techniques, Analytical↗
PubMed1999

An immunoassay for assessment of receptor tyrosine kinase autophosphorylation.

We have developed an immunoassay that can be used to assess autophosphorylation activity of receptor tyrosine kinases, yet does not require the use of synthetic peptide substrates or anti-receptor antibodies. In the assay described here, receptor autophosphorylation and detection take place entirely within the wells of 96 well microplates coated with Protein G and an anti-phosphotyrosine antibody. As the kinase reaction takes place, the antibodies capture the phosphorylated products in situ. Phosphorylation levels of captured receptors are measured by scintillation counting. Assay parameters were validated using A431 cell extracts containing EGF Receptor. The autophosphorylation capture assay is a simple and rapid method which can be adapted for use with robotics for qualitative HTS of potential inhibitors to any tyrosine kinase of interest.

Antibodies↗
PubMed1996

Improving the copy numbers of antibody fragments expressed on the major coat protein of bacteriophage M13.

BACKGROUND: Antibody fragments have been expressed on the major coat protein of filamentous phage using a gene VIII expression system, but with low copy numbers (averaging 0.2 Fab/phage). OBJECTIVES: As a general strategy to increase copy number, the phage vector was optimized by site directed mutagenesis. STUDY DESIGN: One mutation was the introduction of a random six amino acid tether between the heavy chain and pseudo gene VIII to form a phage library. The other mutation was the removal of two cysteine residues which form a disulfide bond between the heavy and light chains. An assay was developed to measure Fab concentrations and used to calculate the average number of copies displayed on phage. RESULTS: Phage libraries containing random tethers were panned, and clones containing a proline rich motif were extracted. Removing the interchain disulfide had a greater effect on copy number and soluble Fab concentrations in the periplasmic space of the bacterial cultures. CONCLUSION: A tenfold increase in the copy number was achieved using the optimized vector. Incorporation of these vector mutations may be a general strategy for optimizing Fab display on the major coat protein of bacteriophage M13.

Bacteriophage M13↗