Biomedical subjects

Seok Jae Lee

Publications and source records attributed to Seok Jae Lee.

6 recordsLinked to original sources

PubMed2006

Protein nanopatterns and biosensors using gold binding polypeptide as a fusion partner.

An efficient strategy for immobilizing proteins on a gold surface was developed by employing the gold binding polypeptide (GBP) as a fusion partner. Using the enhanced green fluorescent protein (EGFP), severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVme), and core streptavidin (cSA) of Streptomyces avidinii as model proteins, specific immobilization of the GBP-fusion proteins onto the gold nanoparticles and generation of protein nanopatterns on the bare gold surface were demonstrated. The GBP-fused SCVme bound to gold nanoparticles successfully interacted with its antibody and showed changes in absorbance and color, allowing efficient diagnosis of SARS-CoV. The fusion proteins could be successfully immobilized on the gold surface by nanopatterning and microcontact printing as examined by atomic force microscopy and surface plasmon resonance analysis. The poly(dimethylsiloxane) microfluidic channels were created on the gold surface and were used for antigen-antibody and DNA-DNA interaction studies. Specific immobilization of GBP-EGFP fusion protein and its interaction with the antibody in the microchannels could be demonstrated. By immobilizing the DNA probe through the use of GBP-fused cSA, specific hybridization of the target DNA prepared from Salmonella could also be achieved. The GBP-fusion method allows immobilization of proteins onto the gold surface without surface modification and in bioactive forms suitable for studying protein-protein, DNA-DNA, and other biomolecular interaction studies. Furthermore, these studies can be carried out in a microfluidic system, which allows high-throughput analysis of biomolecular interactions.

Biological Assay↗

PubMed2005

Micropatterning proteins on polyhydroxyalkanoate substrates by using the substrate binding domain as a fusion partner.

A novel strategy for micropatterning proteins on the surface of polyhydroxyalkanoate (PHA) biopolymer by microcontact printing (microCP) is described. The substrate binding domain (SBD) of the Pseudomonas stutzeri PHA depolymerase was used as a fusion partner for specifically immobilizing proteins on PHA substrate. Enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) fused to the SBD could be specifically immobilized on the micropatterns of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Laser scanning confocal microscopic studies suggested that two fusion proteins were micropatterned in their functionally active forms. Also, antibody binding assay by surface plasmon resonance suggested that protein-protein interaction studies could be carried out using this system.

Adsorption↗

PubMed2005

Selective immobilization of fusion proteins on poly(hydroxyalkanoate) microbeads.

A novel fusion protein system employing the substrate-binding domain (SBD) of poly(hydroxyalkanoate) (PHA) depolymerase was developed for the specific immobilization of proteins on PHA microbeads, and was consequently used for immunoassays. The enhanced green fluorescent protein, red fluorescent protein, and severe acute respiratory syndrome coronavirus envelope protein were used as model proteins, and were selectively and functionally immobilized to the PHA microbeads by fusing them to the SBD. Using this PHA microbead system combined with SBD fusion technology, immunoassays could be successfully carried out.

Flow Cytometry↗

PubMed2004

Micropatterns of spores displaying heterologous proteins.

Micropatterns of Bacillus thuringiensis spores were generated by a combination of surface chemistry, microcontact printing, and spore surface display technique. The outermost layers of B. thuringiensis spores were engineered to present enhanced green fluorescent protein (EGFP), and the biospecific interaction between biotin and streptavidin was utilized to spatially direct the EGFP-presenting spores onto the micropatterned surfaces. The viability of the micropatterned spores was confirmed by the pattern generation of vegetative cells after the germination.

Bacillus thuringiensis↗

PubMed2004

Microarrays of peptides elevated on the protein layer for efficient protein kinase assay.

Peptide microarrays can be used for the high-throughput analysis of protein-peptide interactions. However, current peptide microarrays are rather costly to make and require cumbersome steps of introducing novel polymeric surfaces and/or chemical derivatization of peptides. Here, we report a novel method for manufacturing peptide microarrays by elevating the peptide on the layer of protein by a fusion protein approach. Using two protein kinases and their peptide substrates as examples, we show that elevating peptides on the layer of protein allows sensitive, specific, and efficient detection of peptide-protein interactions without the need for complicated chemical modification of solid supports and peptides. It was found that kinase activity could be detected with as low as 0.09 fmol of kemptide, which is about 1000-fold more sensitive than the 0.1 pmol obtained with other microarray systems. Furthermore, peptides can be produced as fusion proteins by fermentation of recombinant Escherichia coli and thus the expensive peptide synthesis process can be avoided. Therefore, this new strategy will not only be useful in high-throughput and cost-effective screening of kinase substrate peptides but also be generally applicable in studying various protein-peptide interactions.

Binding Sites↗

PubMed2003

Enhanced production of insulin-like growth factor I fusion protein in Escherichia coli by coexpression of the down-regulated genes identified by transcriptome profiling.

The transcriptome profiles of recombinant Escherichia coli producing human insulin-like growth factor I fusion protein (IGF-I(f)) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly down-regulated during the production of IGF-I(f) after induction. Among these down-regulated genes, we rationally selected and coexpressed in E. coli producing IGF-I(f) the prsA gene (encoding a phosphoribosyl pyrophosphate synthetase) and the glpF gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-I(f) could be increased from 1.8 +/- 0.13 (+/- standard deviation) to 4.3 +/- 0.24 g/liter. The volumetric productivity was also increased from 0.36 +/- 0.027 to 0.82 +/- 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance.

Aquaporins↗