EXPRESSION OF PLACENTAL GROWTH HORMONE RELATED PROTEINS

休假训练的总体目标将是合成重组 大鼠胎盘生长激素/催乳素相关蛋白 表征它们对不同生物的结合和生物活性 受体的形式。我的实验室最近发现了一个新的家庭 该研究由美国国立卫生研究院资助，用于研究GHRPs的作用 在胎盘生长过程中。休假的目标是综合 GHRP家族中的单个蛋白质，评估它们各自的能力 结合和激活不同形式的受体，并建立一种 预测孕鼠潜在促生长作用的依据。 第一个特定的目标是产生稳定表达的细胞系 大鼠胎盘GHRPs和催乳素样蛋白B和 C.含有重组蛋白的培养基将被评估为 与天然生长激素和催乳素受体的结合活性，如 以及克隆的受体，这些受体在DR中稳定表达。 凯利的实验室。第一学期的休假将从9月1日开始。 1992年至1993年2月。我在佛罗里达大学的实验室将 随后对重组蛋白进行纯化。 在1992年3-8月期间转染组细胞系。第二 具体目的是对纯化的重组蛋白进行鉴定 利用克隆的长短型的结合和生物活性 受体在CHO细胞中表达。功能将使用以下工具进行评估 β-乳珠蛋白与不同PRL受体基因共转染的研究 启动子-氯霉素乙酰转移酶受体基因 建造。这项工作将在第二届任期内进行。 1993年9月至11月在法国休假。我在佛罗里达的实验室将 随后开始对纯化的重组蛋白进行鉴定 1-8月孕鼠促生长作用的研究 1994年。在休假的第三个学期，从1994年9月到11月， 第三个具体目标将是合成突变形式的重组 使用定点突变的胎盘蛋白。该装订和 突变蛋白的生物活性将被评估为 在凯利博士的实验室里克隆的受体。突变的蛋白质将 随后被提纯并评估其促进生长的活性 佛罗里达大学的几个低出生体重动物模型。

The overall goal of sabbatical training will be to synthesize recombinant rat placental growth hormone/prolactin related protein (GHRPs) and to characterize their binding and biological activities toward different forms of receptors. My laboratory has recently identified a novel family of GHRPs in rat placenta and is funded by NIH to study the role of GHRPs in feto-placental growth. The goal of the sabbatical is to synthesize individual proteins in the GHRP family, evaluate their respective ability to bind and activate different forms of receptors, and to establish a basis for predicting potential growth-promoting effects in pregnant rats. The first specific aim is to generate cell lines which stably express cDNAs for the rat placental GHRPs and the Prolactin-Like-Proteins B and C. Culture media containing recombinant proteins will be evaluated for binding activity to native growth hormone and prolactin receptors, as well as to cloned receptors which have been stably expressed in Dr. Kelly's laboratory. The first term of the sabbatical will be from Sep 1992-Feb 1993. My laboratory at the University of Florida will subsequently purify the recombinant proteins synthesized by the transfected cell lines during the period of Mar-Aug 1992. The second specific aim is to characterize the purified recombinant proteins for binding and biological activities using cloned long and short forms of receptors expressed in CHO cells. Functionality will be evaluated using cotransfection of different PRL receptor cDNAs with a Beta-lactoglobin promoter-chloramphenicol acetyltransfersase (CAT) receptor gene construct. This work will be carried out during the second term of the sabbatical in France from Sep-Nov 1993. My laboratory in Florida will subsequently begin to characterize the purified recombinant proteins for growth-promoting effects in pregnant rats during the period of Jan-Aug 1994. During the third term of the sabbatical, from Sep-Nov 1994, the third specific aim will be to synthesize mutated forms of recombinant placental proteins using site-directed mutagenesis. The binding and biological activities of the mutated proteins will be evaluated toward cloned receptors in Dr. Kelly's laboratory. The mutated proteins will subsequently be purified and evaluated for growth-promoting activity in several animal models of low birth weight at the University of Florida.