B. Hodges NIH Amgen Poster Presentation
1
Recombinant Endothelial NOS Protein Expression to Study Alpha- globin Function Brittany Hodges, Bryan Mott, Ph.D., Hans Ackerman, M.D., D.Phil, Dongying Ma, Ph.D. Sickle Cell Branch, National Heart, Lung, Blood Institute, National Institutes of Health, Bethesda, Maryland Introductio n • Alpha globin is a subunit of hemoglobin, an iron-containing transport protein in red blood cells. In alpha thalassemia, at least 1 of 4 α-globin genes have been deleted. Continued research is needed to determine how α-globin plays a role in blood vessel regulation. 1 • Recently endothelial alpha-globin was found to regulate NO availability via direct interaction with endothelial nitric oxide synthase (eNOS). 2 • To further investigate the mechanism of eNOS/ alpha-globin interaction and its association with blood flow and pressure, recombinant eNOS will be expressed and purified. 1. Restriction enzyme digestion and ligation Statement of Purpose Recombinant protein expression of eNOS oxygenase. Methods • eNOS gene was digested using EcoRΙ and BamHΙ from PUC57 and ligated into PGEX-2T vector • Transformation of eNOS/PGEX-2T to TOP10 competent cells • eNOS/PGEX-2T plasmid extraction from TOP10 cell culture • eNOS/PGEX-2T transformation to E. coli UT5600 Methods (cont.) • Protein expression of eNOS was induced by IPTG in GC Modified Media • Bacterial Cell Lysis of UT5600 to release protein • SDS-PAGE Gel Electrophoresis and Western Blot to check protein expression of eNOS • eNOS purification using GSTrap™ column Results PGEX-2T eNOS/PUC57 plasmid 2. Plasmid extraction from TOP10 3. Cell culture and protein expression Results (cont.) 6. Protein purification by GSTrap™ Summary Conclusions References Acknowledgement s • eNOS oxygenase was subcloned into expression vector PGEX-2T • Recombinant plasmid was transformed to E. coli UT5600 cells • Protein expression was induced by IPTG • Protein was purified by affinity chromatography (GSTrap™) • Western Blot was used to detect the protein purity This internship was completed with funding from the Amgen Scholars program at the NIH. The research was completed under the supervision of Dr. Hans Ackerman and • Recombinant eNOS oxygensase domain was successfully expressed in E. coli. • Purified protein will be used in high-throughput screening for drugs that disrupt eNOS/α-globin interaction and can potentially regulate blood pressure 1."Alpha Thalassemia." Genetics Home Reference. NIH U.S. National Library of Medicine. 2.Straub, Adam C. et al. “Endothelial Cell Expression of Hemoglobin α Regulates Nitric Oxide Signaling.” Nature 491.7424 (2012): 473–477. 1.Sample before loading 2.Flowthrough 3.Washing Buffer 4.Elution Buffer 5.Marker: SeeBlue 1 2 3 4 5 4. Staining with Ponseau S 5. Western Blot using Anti-GST antibody DTT - + DTT - + Regulation of NO signaling by α- globin Transformation of eNOS/PGEX-2T plasmid to TOP10 E. coli
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Transcript of B. Hodges NIH Amgen Poster Presentation
Recombinant Endothelial NOS Protein Expression to Study Alpha-globin FunctionBrittany Hodges, Bryan Mott, Ph.D., Hans Ackerman, M.D., D.Phil, Dongying Ma, Ph.D.
Sickle Cell Branch, National Heart, Lung, Blood Institute, National Institutes of Health, Bethesda, Maryland
Introduction• Alpha globin is a subunit of
hemoglobin, an iron-containing transport protein in red blood cells. In alpha thalassemia, at least 1 of 4 α-globin genes have been deleted. Continued research is needed to determine how α-globin plays a role in blood vessel regulation.1
• Recently endothelial alpha-globin was found to regulate NO availability via direct interaction with endothelial nitric oxide synthase (eNOS).2
• To further investigate the mechanism of eNOS/ alpha-globin interaction and its association with blood flow and pressure, recombinant eNOS will be expressed and purified.
1. Restriction enzyme digestion and ligation
Statement of PurposeRecombinant protein expression of eNOS
oxygenase.
Methods• eNOS gene was digested using EcoRΙ
and BamHΙ from PUC57 and ligated into PGEX-2T vector
• Transformation of eNOS/PGEX-2T to TOP10 competent cells
• eNOS/PGEX-2T plasmid extraction from TOP10 cell culture
• eNOS/PGEX-2T transformation to E. coli UT5600
Methods (cont.)• Protein expression of eNOS was induced by IPTG in GC Modified
Media• Bacterial Cell Lysis of UT5600 to release protein• SDS-PAGE Gel Electrophoresis and Western Blot to check protein
expression of eNOS• eNOS purification using GSTrap™ column
Results
PGEX-2T eNOS/PUC57 plasmid
2. Plasmid extraction from TOP10
3. Cell culture and protein expression
Results (cont.)6. Protein purification by
GSTrap™
Summary
Conclusions
References
Acknowledgements
• eNOS oxygenase was subcloned into expression vector PGEX-2T
• Recombinant plasmid was transformed to E. coli UT5600 cells
• Protein expression was induced by IPTG• Protein was purified by affinity
chromatography (GSTrap™) • Western Blot was used to detect the
protein purity
This internship was completed with funding from the Amgen Scholars program at the NIH. The research was completed under the supervision of Dr. Hans Ackerman and Dr. Dongying Ma.
• Recombinant eNOS oxygensase domain was successfully expressed in E. coli.
• Purified protein will be used in high-throughput screening for drugs that disrupt eNOS/α-globin interaction and can potentially regulate blood pressure
1. "Alpha Thalassemia." Genetics Home Reference. NIH U.S. National Library of Medicine.
2. Straub, Adam C. et al. “Endothelial Cell Expression of Hemoglobin α Regulates Nitric Oxide Signaling.” Nature 491.7424 (2012): 473–477.
1.Sample before loading
2.Flowthrough3.Washing Buffer4.Elution Buffer5.Marker: SeeBlue
1 2 3 4 5
4. Staining with Ponseau S 5. Western Blot using Anti-GST antibodyDTT
- +
DTT - +
Regulation of NO signaling by α-globin
Transformation of eNOS/PGEX-2T plasmid to
TOP10 E. coli
