Biomedical subjects

Y G Mikawa

Publications and source records attributed to Y G Mikawa.

4 recordsLinked to original sources

PubMed1997

Efficient epitope mapping by bacteriophage lambda surface display.

A bacteriophage lambda surface expression system, lambda foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase 1 to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries.

Amino Acid Sequence↗

PubMed1996

Surface display of proteins on bacteriophage lambda heads.

We have developed plasmid and phage vectors for the display of foreign proteins on the surface of bacteriophage lambda capsid by modifying the D gene which encodes the major head protein gpD. The vectors have multiple cloning sites, and permit colour selection and conditional chain termination for recombinants. Displayed proteins can be fused to either the N or C terminus of gpD by a peptide linker. The conditional chain termination scheme, via a host Escherichia coli suppressor activity, allows the fusion and assembly of homomultimeric proteins as well as control of the number of fusion proteins per phage particle. We have successfully displayed beta-lactamase, IgG-binding domains of the Staphylococcus aureus protein A, and beta-galactosidase by cloning the genes into the vector. The constructs express functionally active proteins fused to gpD that assemble into phage particles. These results suggest that gpD may be fused to many other peptides and proteins at their N or C terminus and the fusion products may be accessible on the surface of bacteriophage lambda particles.

Amino Acid Sequence↗

PubMed1995

A model for transmembrane signalling by the aspartate receptor based on random-cassette mutagenesis and site-directed disulfide cross-linking.

Extracellular information is transduced by transmembrane receptors into the inside of the cell across a membrane barrier. To understand the molecular basis of transmembrane signalling, we replaced the transmembrane segment 2 (TM2) of the Escherichia coli aspartate receptor, Tar, with random sequences that are 21 amino acid residues in length and consist of Arg, Gly, Ser, Cys, Val, Leu, Ile and Phe at each position. From this ensemble for recombinant molecules, functional receptors were recovered as clones that could bind aspartate and transmit a signal to the intracellular domain. Restricted average hydrophobicity values were observed for functional transmembrane domains, and support the observation that transmembrane segments typically have hydrophobicity values greater than 1.6. However, non-functional transmembrane domains with greater hydrophobicity than 1.6 indicate that hydrophobicity is not a sole determinant for its function. Fourier transform analysis of the functional TM2 sequences suggests that the transmembrane segment has an alpha-helical structure with three distinct faces. Cross-linking of the faces to transmembrane segment 1 (TM1) mimics the "locked" signalling phenotypes of the wild-type receptor. The results are consistent with a model in which TM2 rotates in the plane of the lipid bilayer, and the rotation becomes locked at one face of the alpha-helix in the presence of attractant and at another face in the presence of repellent. This dynamic movement of the transmembrane domain may be a common signalling mechanism of homologous membrane receptor molecules such as the insulin receptor. Random-cassette mutagenesis and disulfide cross-linking provide powerful strategies for examining the structure and function of transmembrane segments.

Amino Acid Sequence↗

PubMed1993

A bacteriophage T7-based expression vector, pBT7, with color selection for the recombinant.

A bacterial expression plasmid, pBT7, based on a bacteriophage T7 RNA polymerase/promoter system, has been devised for positive color selection of clones containing an insert. The T7 gene 10 promoter and its N-terminal portion, 89 nucleotides in length, have been amplified by the polymerase chain reaction and cloned into the multiple cloning site of plasmid pUC18 in the opposite direction to lacZ'. This insertion does not inactivate the lacZ alpha-peptide activity. Hence, bacteria carrying pBT7 without an insert form blue colonies on an indicator plate, while bacteria carrying recombinant clones with an insert appear as white colonies. Using pBT7, part of the Caenorhabditis elegans unc-13 gene product has been overproduced at a high level, approaching 50% of total Escherichia coli protein.

Animals↗