CORE C. RECOMBINANT HBA PRODUCTION FOR IN VIVO TOXICITY TESTING PI: John S. Olson, Rice University DESCRIPTION: ABSTRACT: All three projects in this program require large amounts of recombinant Hb mutants to evaluate the causes of acellular Hb toxicity in simple cellular systems, organs, and whole animals. The work with perfused hearts and whole animal exchange transfusion models requires scaling up our current production of -0.5 to 1 g of rHb per week by at least 10-fold. We also need to achieve complete removal of endotoxin (LPS), which requires dedicated purification facilities to remove all traces of E. coli proteins and membranous materials from final preparations and special packaging. These goals will be achieved by setting up a rHb prodUctioh facility at Rice University, which will bper'ate cohtihuously to pr'bvide the LPS- free rHb solutions for Projects 1, 2, and 3. A full time Ph.D. staff scientist will run the facility with the help of a research assistant and other members of the Pi's laboratory, who are part of Project 2. A 30 L B.Braun Biostat C+ 30 L will be purchased and dedicated solely to rHb production and be run in parallel with our existing 20L Biostat C fermentor, whenever possible.
Our specific aims are to: (1) produce at least 6 g of LPS-free recombinant Hb samples per week or ^ 300 grams per year using current expression and production technology;and (2) enhance these production yields 2 to 3-fold by integrating the Plesiomonas shigelloides heme transport system (heme utilization genes, hug) and alpha hemoglobin stabilizing protein (AHSP) into the expression protocol to facilitate holoprotein assembly and to increase the efficiency of purification by decreasing protein miss-folding and preventing the accumulation of hemin degradation products during bacterial growth. PERFOMANCE

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL110900-02
Application #
8517811
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
2
Fiscal Year
2013
Total Cost
$217,770
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Kassa, Tigist; Strader, M B; Nakagawa, Akito et al. (2017) Targeting ?Cys93 in hemoglobin S with an antisickling agent possessing dual allosteric and antioxidant effects. Metallomics 9:1260-1270
Alayash, Abdu I (2017) Mechanisms of Toxicity and Modulation of Hemoglobin-Based Oxygen Carriers (HBOCs). Shock :
Martucci, Alexandre Fabricio; Abreu Martucci, Ana Carolina Carvalho Ferreira; Cabrales, Pedro et al. (2017) Acute kidney function and morphology following topload administration of recombinant hemoglobin solution. Artif Cells Nanomed Biotechnol 45:24-30
Ao-Ieong, Eilleen S Y; Williams, Alexander; Jani, Vivek et al. (2017) Cardiac function during resuscitation from hemorrhagic shock with polymerized bovine hemoglobin-based oxygen therapeutic. Artif Cells Nanomed Biotechnol 45:686-693
Jana, Sirsendu; Meng, Fantao; Hirsch, Rhoda E et al. (2017) Oxidized Mutant Human Hemoglobins S and E Induce Oxidative Stress and Bioenergetic Dysfunction in Human Pulmonary Endothelial Cells. Front Physiol 8:1082
Draganski, Andrew R; Friedman, Joel M; Ludescher, Richard D (2017) Solvent-Slaved Dynamic Processes Observed by Tryptophan Phosphorescence of Human Serum Albumin. Biophys J 112:881-891
Meng, Fantao; Alayash, Abdu I (2017) Determination of extinction coefficients of human hemoglobin in various redox states. Anal Biochem 521:11-19
Bissé, Emmanuel; Schaeffer-Reiss, Christine; Van Dorsselaer, Alain et al. (2017) Hemoglobin Kirklareli (? H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding. J Biol Chem 292:2542-2555
Zimmerman, Robert; Tsai, Amy G; Salazar Vázquez, Beatriz Y et al. (2017) Posttransfusion Increase of Hematocrit per se Does Not Improve Circulatory Oxygen Delivery due to Increased Blood Viscosity. Anesth Analg 124:1547-1554
Jani, Vivek P; Jelvani, Alborz; Moges, Selamawit et al. (2017) Polyethylene Glycol Camouflaged Earthworm Hemoglobin. PLoS One 12:e0170041

Showing the most recent 10 out of 93 publications