Biomedical subjects

D J Rodi

Publications and source records attributed to D J Rodi.

6 recordsLinked to original sources

PubMed2006

Bacterial expression strategies for human angiogenesis proteins.

We outline an expression strategy using Escherichia coli to obtain soluble components of a selected group of human proteins implicated in angiogenesis. These targets represent a heterogeneous group of proteins which for expression purposes were separated into cytoplasmic and helical membrane protein categories. Target selection was refined using a bioinformatic approach to generate a list of 50 experimental targets. A group consisting of forty-four cytoplasmic and signal-containing protein targets were amplified and cloned into multiple expression vectors. For this target category, we obtained 48% soluble expression products. In addition, we used a domain expression approach for six high molecular weight proteins predicted to contain membrane spanning helices to obtain soluble domain products. These results validate the utility of a bioinformatically driven high throughput approach to increase the number of soluble proteins or protein domains which can be used for multiple downstream applications.

Angiogenesis Inducing Agents↗

PubMed2004

Wide-angle X-ray solution scattering as a probe of ligand-induced conformational changes in proteins.

A chemical genetics approach to functional analysis of gene products utilizes high-throughput target-based screens of compound libraries to identify ligands that modulate the activity of proteins of interest. Candidates are further screened using functional assays designed specifically for the protein--and function--of interest, suffering from the need to customize the assay to each protein. An alternative strategy is to utilize a probe to detect the structural changes that usually accompany binding of a functional ligand. Wide-angle X-ray scattering from proteins provides a means to identify a broad range of ligand-induced changes in secondary, tertiary, and quaternary structure. The speed and accuracy of data acquisition, combined with the label-free targets and binding conditions achievable, indicate that WAXS is well suited as a moderate-throughput assay in the detection and analysis of protein-ligand interactions.

Alcohol Dehydrogenase↗

PubMed2001

Identification of small molecule binding sites within proteins using phage display technology.

Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

Bacteriophages↗

PubMed1999

Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein.

A random library of phage displayed peptides was screened for binding to a biotinylated derivative of paclitaxel (Taxol). Affinity-selected peptides were analyzed for similarity to human proteins. There was no significant similarity between the paclitaxel-selected peptides and tubulin. However, a subset of the peptides was identified that exhibits significant similarity to a non-conserved region of the anti-apoptotic human protein Bcl-2: ELISA assays confirmed binding of paclitaxel to Bcl-2, and circular dichroism spectroscopy demonstrated that a substantial conformational change accompanies this binding. In vivo, treatment with paclitaxel has been shown to lead to Bcl-2 inactivation with concomitant phosphorylation of residues in a disordered, regulatory loop region of the protein. Similarity between paclitaxel-selected peptides and this loop region implicate these residues in drug binding, and suggest that the apoptotic action of paclitaxel may involve the binding of paclitaxel to Bcl-2. These results demonstrate that peptides displayed on the surface of bacteriophage particles can mimic the ligand-binding properties of disordered regions of proteins.

Amino Acid Sequence↗

PubMed1999

Phage-display technology--finding a needle in a vast molecular haystack.

Screening of phage-displayed libraries of proteins and peptides has, for nearly a decade, proven to be a highly effective method for finding much needed 'needles' in a vast molecular 'haystack'. Over the past year, it has been used to solve an increasing diversity of problems, including identification of binding motifs for much smaller targets and the use of novel screening methods to identify chemical activities.

Bacteriophage M13↗

PubMed1999

Similarity between the sequences of taxol-selected peptides and the disordered loop of the anti-apoptotic protein, Bcl-2.

The anti-cancer drug taxol is known to bind to and induce the polymerization of tubulin and has recently been shown to bind to the anti-apoptotic protein Bcl-2, but not to its homolog, Bcl-XL. Libraries of random peptides displayed on the surface of a bacteriophage were screened to select those exhibiting affinity for taxol. The sequences of these peptides were compared to sequences of proteins involved in mitosis and apoptosis. No significant similarities were detected between the sequences of tubulins and the taxol-selected peptides. However, a high level of similarity exists between the selected peptides and the disordered loop of Bcl-2. Conversely, there was little similarity between the sequences of the selected peptides and Bcl-XL. These results indicate that peptides displayed on the surface of a bacteriophage can mimic the ligand-binding behavior of a disordered protein loop and that comparison of the sequences of affinity-selected peptides with protein sequences can be predictive for ligand binding.

Amino Acid Sequence↗