No abstract

Apolipoprotein A‐I (apoA‐I) is the most abundant protein in high‐density lipoprotein, an anti‐antherogenic complex responsible for reverse cholesterol transport. Human apoA‐I is a 243 amino acid protein comprised of two domains, a helix bundle N‐terminal domain, and an unstructured C‐terminal (CT) domain comprising residues 179‐243 which adopts a helical conformation when lipid bound. To better understand the function of the CT apoA‐I, a novel strategy was employed to produce large quantities of this small fragment using a recombinant expression system. The strategy required the introduction of a methionine residue in front of the CT domain to be able to cleave with cyanogen bromide and isolate the desired fragment. However, apoA‐I contains three methionine residues making it difficult to cleave and isolate the correct fragment. Therefore, a new construct was engineered by combining CT apoA‐I with apolipophorin III (apoLp‐III), an insect apolipoprotein that lacks methionine. A unique methionine was then introduced by site‐directed mutagenesis between apoLp‐III and CT apoA‐I. The chimeric protein was expressed in E. coli BL21 cells, purified by nickel affinity chromatography, and cleaved by cyanogen bromide. SDS‐PAGE revealed the presence of three distinct bands at 7 kDa (CT apoA‐I), 18 kDa (apoLp‐III), and a minor band at 26 kDa of uncleaved protein. Then, reversed‐phase HPLC was employed to isolate the 7 kDa fragment. SDS‐PAGE of the first peak eluted from the C8 column confirmed isolation of CT apoA‐I. This novel expression system allows for production of significant quantities of CT apoA‐I needed for the analysis to understand how this important part of apoA‐I functions.This project was supported by NIH/NIGMS GM089564 and GM008074.

Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179–243 of apoA-I, with a unique a methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification. The chimera was expressed in E. coli, purified by Ni-affinity chromatography, and cleaved by cyanogen bromide. SDS-PAGE revealed the presence of three proteins with masses of 7 kDa (CT-apoA-I), 18 kDa (apoLp-III), and a minor 26 kDa band of uncleaved chimera. The digest was reloaded on the Ni-affinity column to bind apoLp-III and uncleaved chimera, while CT-apoA-I was washed from the column and collected. Alternatively, CT-apoA-I was isolated from the digest by reversed-phase HPLC. CT-apoA-I was α-helical, highly effective in solubilizing phospholipid vesicles and disaggregating LPS micelles. However, CT-apoA-I was less active compared to full-length apoA-I in protecting lipolyzed low density lipoproteins from aggregating, and disrupting phosphatidylglycerol bilayer vesicles. Thus the novel expression system produced mg quantities of functional CT-apoA-I, facilitating structural and functional studies of this critical domain of apoA-I.

Background: Under normal conditions, endothelial cells (ECs) govern blood flow dynamics including providing a barrier between blood and tissue and regulating platelet aggregation and thrombin generation in the bloodstream. In turn, blood components, primarily platelets and coagulation factors such as thrombin, regulate EC barrier integrity. The breakdown of EC barrier function is a hallmark of a variety of vascular diseases. In sepsis, for example, the dysfunction of vascular ECs has been correlated with poorer outcomes due to hemorrhage and multi-organ failure associated with consumption of platelets and coagulation factors into clots within the microcirculation, a condition termed disseminated intravascular coagulation (DIC). Aim: Develop an endothelialized flow chamber to study the platelet-endothelium interface. Methods and Results: We developed a 3D-chamber with a perfuseable cylindrical microvessel embedded in an extracellular matrix (ECM) material. This model allows for the study of the role of thrombin generation and platelet aggregation in endothelial barrier leak development and repair in healthy as well as inflamed microvessels. Incorporation of subendothelial matrix proteins in these 3D-microvessel devices expands the capacity of the microfluidic studies to investigate blood cell extravasation and enables the control of physical parameters such as transmural pressure and interstitial flow through the ECM. Conclusion: This model may provide insight into the pathophysiology of different disease states and serve as an expedient platform for therapy design and testing. The platelet-endothelium interface under shear flow. Diagram ( A ) and an experimental prototype ( B ) of a 3D-perfuseable device. Microvessel phenotype (following treatment with vehicle or 10 ng/mL TNFα) pre- and post- perfusion with recalcified whole blood for 33 min as visualized by differential interference contrast, DIC, ( C ) and fluorescence microscopy ( D ).