CORE--FACILITY FOR RECOMBINANT AND MODIFIED SICKLE HEMOGLOBINS

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DESCRIPTION (provided by applicant): The major goal of the Scientific Core is to provide adequate quantities of recombinant sickle hemoglobins with varying substitutions as well as chemically modified hemoglobins (including cross linked hemoglobins) to the individual projects. The methods to produce these are already in place and have been used during the present grant period. In addition, improvements have been made in the procedures for expressing recombinant hemoglobins so that the yields (75- 100 mg of purified Hb per fermentation) have been significantly improved. Fermentation procedures have been modified to shorten them (about 5 days) successive runs now practical and will aid in multiplying the above yields as necessary to meet needs of individual projects. A second fermenter (60 liters) will be set up in addition to the unit now being used (20 liters). The specific aims of the Scientific Core will be: Recombinant HbS Mutants - These will be of the general type a2x sB2x meaning that mutations can be introduced on either a- or B-chains of HbS or both chains. These can be at multiple mutation sites per chain, as already demonstrated in our publications. Mutation sites will be identified after consultation with Dr. Robert Josephs or any of the other P.I.'s. The nature of the replacement (charged, uncharged, hydrophilic, hydrophobic) will be determined after consultations with other project leaders. Crosslinking - Two crosslinkers are being used in our laboratory (DIBS and DBBF). These react at different sites on Hb and procedures have been developed to purify and characterize the desired products since usually more than one site is reacted. The aim will be to prepare stable crosslinked (Asymmetric) tetrameric hybrids of HbS-HbA, HbS-HbS-HbF, HbS-HbA2 and others as the need presents itself.