Browsing by Author "Chen, Zhilei"

Now showing 1 - 4 of 4

A Novel Technology for Engineering Binders to Membrane Proteins
Microbial Pathogenesis And Immunology; TAMHSC; National Institutes of Health
Antibodies have been coined the ‘magic bullets’ against many human diseases. However, there remains significant challenges in the engineering of antibodies targeting multi-pass membrane proteins, which encompass a large number of therapeutic targets such as cell surface receptors and the ion channel proteins. The difficulty in engineering binders to membrane proteins stems from the limitation of the current in vitro selection/panning technologies, such as phage display, which require highly purified target protein. Unfortunately, membrane proteins are often refractory to purification due to their dependence on the cell membrane for proper folding and activity. Currently, there is no effective in vitro technology for the discovery/engineering of binders to multi-pass membrane proteins. The overall goal of this study is to develop a novel technology – SMURF (Simple proxiMity coUpled mRNA display) – for engineering protein binders to protein targets on the cell surface, thus bypassing the need to purify the target protein. SMURF combines mRNA display with the proximity-assisted-DNA-assembly phenomenon and, unlike conventional panning in which all binders to a solid support are enriched, SMURF fosters the enrichment of binders only to a desired target protein on the cell surface. In Aim 1, we will demonstrate the SMURF principle using oligonucleotides and optimize the primer sequences. Aim 2 will establish the SMURF enrichment of a model protein in a mixture of non-target proteins in solution. Finally, in Aim 3, a model protein will be displayed on the mammalian cell surface and a library of binders will be screened to demonstrate and quantify the whole-cell SMURF enrichment efficiency. The successful completion of this study will establish a novel technology for facile discovery/engineering of binders to whole-cell-displayed membrane proteins and should greatly expand the repertoire of drug targets amenable to therapeutic intervention.
A Novel Technology for Engineering Binders to Membrane Proteins
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/217; National Institutes of Health
Antibodies have been coined the ‘magic bullets’ against many human diseases. However, there remains significant challenges in the engineering of antibodies targeting multi-pass membrane proteins, which encompass a large number of therapeutic targets such as cell surface receptors and the ion channel proteins. The difficulty in engineering binders to membrane proteins stems from the limitation of the current in vitro selection/panning technologies, such as phage display, which require highly purified target protein. Unfortunately, membrane proteins are often refractory to purification due to their dependence on the cell membrane for proper folding and activity. Currently, there is no effective in vitro technology for the discovery/engineering of binders to multi-pass membrane proteins. The overall goal of this study is to develop a novel technology – SMURF (Simple proxiMity coUpled mRNA display) – for engineering protein binders to protein targets on the cell surface, thus bypassing the need to purify the target protein. SMURF combines mRNA display with the proximity-assisted-DNA-assembly phenomenon and, unlike conventional panning in which all binders to a solid support are enriched, SMURF fosters the enrichment of binders only to a desired target protein on the cell surface. In Aim 1, we will demonstrate the SMURF principle using oligonucleotides and optimize the primer sequences. Aim 2 will establish the SMURF enrichment of a model protein in a mixture of non-target proteins in solution. Finally, in Aim 3, a model protein will be displayed on the mammalian cell surface and a library of binders will be screened to demonstrate and quantify the whole-cell SMURF enrichment efficiency. The successful completion of this study will establish a novel technology for facile discovery/engineering of binders to whole-cell-displayed membrane proteins and should greatly expand the repertoire of drug targets amenable to therapeutic intervention.
Internal Toxin Neutralizer for Treating STEC-infection
Microbial Pathogenesis And Immunology; TAMHSC; DHHS-NIH-National Institute of Allergy and Infectious Diseases
Abstract The Shiga toxin-producing E. coli (STEC) is the most common cause of bloody diarrhea and afflicts an estimated 73,000 people in the US annually, causing significant morbidity. The most recent and largest STEC outbreak occurred in Germany in 2011, affecting >3,800 people, including 54 deaths. Currently there is no effective treatment for STEC infection. The pathology of STEC infection derives from two exotoxins – Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) – that are secreted by STEC in the gut. Although antibiotic treatment can reduce the load of STEC, it also augments Shiga toxin release, leading to increased risk of developing the more serious hemolytic uremic syndrome (HUS) and kidney failure (up to 25%). Consequently, the CDC recommends that antibiotics not be used in STEC patients and that only supportive therapy (e.g. oral and i.v. fluid, pain control) be used. Although anti-toxin antibodies have been identified, the inability of antibodies to cross the cell membrane renders them powerless against toxins already absorbed by the host cells, limiting their clinical application. We hypothesize that a cytosol-accessible anti-toxin should be able to neutralize both extracellular and intracellular Shiga toxin, leading to a much-prolonged therapeutic window and better therapeutic efficacy. The overall goal of this study is to engineer a panel of intracellular toxin neutralizers (ITNs) against Shiga toxin 2 (Stx2). As a scaffold for the proposed ITN, we will use a designed ankyrin repeat protein (DARPin). DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity and possess low immunogenicity. In this project, we will first isolate DARPins that bind and neutralize Stx2 (Aim 1). Concurrently, we will screen a panel of cell-penetrating peptides (CPPs) for their ability to transport ITNs into cells (Aim 2). In Aim 3, we will assemble anti-Stx2 ITNs using the best anti-Stx2 DARPin and CPP and evaluate the therapeutic potential of these anti-Stx2 ITNs in vitro and in vivo. The approach of using ITN to combat toxins in circulation offers a new paradigm for the treatment of both STEC and non-STEC bacterial infections.
Internal Toxin Neutralizer for Treating STEC-infection
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/217; DHHS-NIH-National Institute of Allergy and Infectious Diseases
The Shiga toxin-producing E. coli (STEC) is the most common cause of bloody diarrhea and afflicts an estimated 73,000 people in the US annually, causing significant morbidity. The most recent and largest STEC outbreak occurred in Germany in 2011, affecting >3,800 people, including 54 deaths. Currently there is no effective treatment for STEC infection. The pathology of STEC infection derives from two exotoxins – Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) – that are secreted by STEC in the gut. Although antibiotic treatment can reduce the load of STEC, it also augments Shiga toxin release, leading to increased risk of developing the more serious hemolytic uremic syndrome (HUS) and kidney failure (up to 25%). Consequently, the CDC recommends that antibiotics not be used in STEC patients and that only supportive therapy (e.g. oral and i.v. fluid, pain control) be used. Although anti-toxin antibodies have been identified, the inability of antibodies to cross the cell membrane renders them powerless against toxins already absorbed by the host cells, limiting their clinical application. We hypothesize that a cytosol-accessible anti-toxin should be able to neutralize both extracellular and intracellular Shiga toxin, leading to a much-prolonged therapeutic window and better therapeutic efficacy. The overall goal of this study is to engineer a panel of intracellular toxin neutralizers (ITNs) against Shiga toxin 2 (Stx2). As a scaffold for the proposed ITN, we will use a designed ankyrin repeat protein (DARPin). DARPins represent a versatile class of binding proteins that have been engineered to bind diverse targets with up to picomolar affinity and possess low immunogenicity. In this project, we will first isolate DARPins that bind and neutralize Stx2 (Aim 1). Concurrently, we will screen a panel of cell-penetrating peptides (CPPs) for their ability to transport ITNs into cells (Aim 2). In Aim 3, we will assemble anti-Stx2 ITNs using the best anti-Stx2 DARPin and CPP and evaluate the therapeutic potential of these anti-Stx2 ITNs in vitro and in vivo. The approach of using ITN to combat toxins in circulation offers a new paradigm for the treatment of both STEC and non-STEC bacterial infections.