Characterization of Lipid-Anchored Inhibitor Rulers as a Measure of Enzyme Topography in Coagulation Enzyme, Factor X
Location
Georgia Campus
Start Date
1-5-2013 2:00 PM
End Date
1-5-2013 4:00 PM
Description
Background: The blood coagulation cascade (BCC) is activated under various circumstances. An injury is a good example of how the process begins. This system is a tightly regulated series of events. The enzymes involved assemble with their respective cofactors on lipid membranes to reach their full pro-coagulant complex potential. Each of these factors is part of the intrinsic pathway and both are required for the activation of factor X. Factor Xa is a very important part of the blood coagulation cascade because its activation is primarily responsible for thrombin generation. Factor Xa, anchored in the plasma membrane, forms a complex with other proteins for example, Factor V. The topography of the Factor Xa alone, and in complex with other factors on the cell surface is poorly understood. This study will focus particularly on the activated form of factor X (fXa).This study will focus particularly on the activated form of factor X (fXa). FX is of the utmost importance in coagulation because it is integral in the generation of thrombin from prothrombin. Kunitz-type protein inhibitors (KPI) are globulin proteins which inhibit trypsin much like the ones that regulate FX. Basic pancreatic trypsin inhibitor (BPTI) is the kunitz-protein inhibitor of particular interest in this study. The goal is to gain understanding of the topography of fXa in the presence of BPTI. More specifically, we will examine the range of reactive heights of the blood clotting factor X/BPTI complex above the membrane surface.
Methods: Previous studies suggest that the height and topology of the active site of FXa above the plasma membrane is of importance to its function. Thus, we would like to generate a series of lipid-anchored ‘inhibitor rulers’ to measure the height of the FXa complex above the plasma membrane. A construct encoding BPTI fused with a bacterial lipid-anchor consensus site (LAGC) separated by ten linker sequences (a total of 75Å) is being built. More specifically, a segment coding for an LAGC leader sequence and EA3K linker sequences is being directionally cloned into pET11d E. coli DNA. Oligonucleotides were designed encoding the EA3K linker repeat and are being introduced into the constructed pET11d plasmid via the engineered restriction endonuclease sites existing between the LAGC sequence and the first part of the BPTI sequence. Next, these lipid-anchored inhibitor rulers will be cloned, expressed, and purified using a series of affinity chromatography steps. Specifically, we will immobilize the enzyme trypsin to the solid support, and use this to separate and purify our newly generated inhibitor product. Once the ruler has been constructed, we will use it to determine the height above the cell membrane for FXa, individually and in the context of their pro-coagulant complexes. Using an equilibrium-based chromogenic enzyme assay, we will measure the rates and extent of inhibition for both FXa and the FXa/pro-coagulant complex. The long-term goal will be to change the length of the ruler and measure the effect on inhibition.
Conclusions: These sequences have been introduced to the plasmid DNA and ligation experiments using a T4 DNA ligase at a 3:1 insert to vector ratio are taking place. Upon ligation, this DNA will be transformed into E. coli DH5α competent cells. Preliminary tests are being done to confirm any colonies with the proper sequence. If any colonies are confirmed preliminarily, they will be sent for DNA sequencing. Upon sequencing confirmation, we will transform the plasmid into an expression strain and begin phase 2 of the project.
Characterization of Lipid-Anchored Inhibitor Rulers as a Measure of Enzyme Topography in Coagulation Enzyme, Factor X
Georgia Campus
Background: The blood coagulation cascade (BCC) is activated under various circumstances. An injury is a good example of how the process begins. This system is a tightly regulated series of events. The enzymes involved assemble with their respective cofactors on lipid membranes to reach their full pro-coagulant complex potential. Each of these factors is part of the intrinsic pathway and both are required for the activation of factor X. Factor Xa is a very important part of the blood coagulation cascade because its activation is primarily responsible for thrombin generation. Factor Xa, anchored in the plasma membrane, forms a complex with other proteins for example, Factor V. The topography of the Factor Xa alone, and in complex with other factors on the cell surface is poorly understood. This study will focus particularly on the activated form of factor X (fXa).This study will focus particularly on the activated form of factor X (fXa). FX is of the utmost importance in coagulation because it is integral in the generation of thrombin from prothrombin. Kunitz-type protein inhibitors (KPI) are globulin proteins which inhibit trypsin much like the ones that regulate FX. Basic pancreatic trypsin inhibitor (BPTI) is the kunitz-protein inhibitor of particular interest in this study. The goal is to gain understanding of the topography of fXa in the presence of BPTI. More specifically, we will examine the range of reactive heights of the blood clotting factor X/BPTI complex above the membrane surface.
Methods: Previous studies suggest that the height and topology of the active site of FXa above the plasma membrane is of importance to its function. Thus, we would like to generate a series of lipid-anchored ‘inhibitor rulers’ to measure the height of the FXa complex above the plasma membrane. A construct encoding BPTI fused with a bacterial lipid-anchor consensus site (LAGC) separated by ten linker sequences (a total of 75Å) is being built. More specifically, a segment coding for an LAGC leader sequence and EA3K linker sequences is being directionally cloned into pET11d E. coli DNA. Oligonucleotides were designed encoding the EA3K linker repeat and are being introduced into the constructed pET11d plasmid via the engineered restriction endonuclease sites existing between the LAGC sequence and the first part of the BPTI sequence. Next, these lipid-anchored inhibitor rulers will be cloned, expressed, and purified using a series of affinity chromatography steps. Specifically, we will immobilize the enzyme trypsin to the solid support, and use this to separate and purify our newly generated inhibitor product. Once the ruler has been constructed, we will use it to determine the height above the cell membrane for FXa, individually and in the context of their pro-coagulant complexes. Using an equilibrium-based chromogenic enzyme assay, we will measure the rates and extent of inhibition for both FXa and the FXa/pro-coagulant complex. The long-term goal will be to change the length of the ruler and measure the effect on inhibition.
Conclusions: These sequences have been introduced to the plasmid DNA and ligation experiments using a T4 DNA ligase at a 3:1 insert to vector ratio are taking place. Upon ligation, this DNA will be transformed into E. coli DH5α competent cells. Preliminary tests are being done to confirm any colonies with the proper sequence. If any colonies are confirmed preliminarily, they will be sent for DNA sequencing. Upon sequencing confirmation, we will transform the plasmid into an expression strain and begin phase 2 of the project.