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 具有不同取代基的血红蛋白以及化学修饰的血红蛋白(包括 交叉连接的血红蛋白)与各个项目相关联。生产这些产品的方法已经 已到位，并已在本赠款期间使用。此外，改进还包括 在表达重组血红蛋白的过程中进行了改进，以使产率(75- 每次发酵100毫克纯化的Hb)已显著提高。发酵 程序已被修改，以缩短连续运行时间(约5天)，现已可行 并将在必要时帮助提高上述收益率，以满足个别项目的需要。一个 在目前使用的发酵罐(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 通用型a2x sB2x意味着突变可以在A-链或B-链上引入 HBS或两者兼而有之。这些基因可能位于每条链上的多个突变位点，就像已经做过的那样 在我们的出版物中得到了展示。将在咨询后确定突变地点 罗伯特·约瑟夫斯博士或任何其他私家侦探S。更换的性质(指控， 不带电、亲水、疏水)将在与其他项目协商后确定 领袖们。交联剂-我们实验室使用了两种交联剂(DIBS和DBBF)。这些反应发生在Hb上的不同部位，由于通常有不止一个部位发生反应，因此已开发出提纯和鉴定所需产物的程序。其目的将是根据需要制备稳定的HBS-HBA、HBS-HBS-HBF、HBS-HbA2和其他的交联型(不对称)四聚体杂化。

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.