CORE--FACILITY FOR RECOMBINANT AND MODIFIED SICKLE HEMOGLOBINS

描述（由申请人提供）： 科学核心的主要目标是提供足够数量的重组镰刀 具有不同取代的血红蛋白以及化学修饰的血红蛋白（包括 交联血红蛋白）到各个项目。生产这些的方法已经 在本补助期内已使用。此外，改进措施还 在用于表达重组血红蛋白的程序中进行，使得产率（75- 每次发酵100 mg纯化Hb）的能力得到了显著提高。发酵 程序已被修改，以缩短它们（约5天），连续运行现在是可行的 并将有助于增加上述产量，以满足个别项目的需要。一 除了现在使用的单元（20升）之外，还将设置第二个发酵罐（60升）。 科学核心的具体目标将是：重组HbS突变体-这些将是 一般类型a2 x sB 2x意味着突变可以引入到 HbS或两条链。这些可以在每条链的多个突变位点，如已经 在我们的出版物中。突变位点将在咨询 博士罗伯特·约瑟夫或其他私家侦探's.更换的性质（收费， 不带电、亲水性、疏水性）将在与其他项目协商后确定 领导人的人选信号 交联-我们实验室使用两种交联剂（DIBS和DBBF）。这些在Hb上的不同位点反应，并且已经开发了纯化和表征所需产物的程序，因为通常不止一个位点反应。目的将是制备HbS-HbA、HbS-HbS-HbF、HbS-HbA 2和其他需要的稳定交联（不对称）四聚体杂合物。

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.