PROTEIN MICROCHEMISTRY SHARED INSTRUMENTATION FACILITY

我们希望建立一种最先进的分析蛋白质微量化学 设施。这将由一个气相定序器及其附属设备组成。 氨基酸分析仪，蛋白质和多肽纯化的高效液相色谱仪，以及 多肽合成设备。带有气相自动测序仪 氨基酸序列分析可以用非常少量的 材料(小于100皮摩尔)。对于一种典型的蛋白质，这与 到5-10微克。这些数量是由单克隆体产生的 抗体亲和层析或制备性二维凝胶 在用户组的各个实验室进行电泳法。建议数 设备将补充大学现有的技术资源 包括寡核苷酸合成器、细胞杂交瘤实验室和 透射电子显微镜设备。它将为积极的研究服务 DNA和c DNA克隆，定点突变，调控 基因表达，以及蛋白质的结构和功能表征。 除了每台机器的单独应用之外，特定的 这些设施的特点是，它们可以在 连词。例如，部分氨基酸序列将用于 确定寡核苷酸杂交探针的可能序列， 这些探针依次用来分离编码最初的 蛋白。相反，从DNA序列可以毫不含糊地推断出 氨基酸序列。与该序列的部分对应的多肽可以 被构建，偶联到载体蛋白上，然后用于生成 抗血清或单抗。这些抗体可以用来 通过刺激或阻断已知的蛋白质来表征蛋白质的功能 功能。最后，可以使用抗体来鉴定新的基因产物。 针对合成的与开放阅读框架相对应的多肽 通过DNA序列分析发现。我们设想了这些技术 相互补充和现有的分子生物学专业知识， 生物化学、免疫学和肿瘤生物学。他们将允许一大笔 解决临床许多基本问题的研究人员数量 和基础科学。

We wish to establish a state-of-the-art Analytical Protein Microchemistry Facility. This will consist of a gas-phase sequenator and its attendant HPLC amino acid analyzer, an HPLC for protein and peptide purification, and peptide synthesis equipment. With the gas-phase sequenator, automated amino acid sequence analysis can be performed with very small amounts of material (less than 100 picomoles). For a typical protein this corresponds to 5-10 micrograms. These amounts are being produced by monoclonal antibody affinity chromatography or preparative two-dimensional gel electrophoresis in various laboratories of the user group. The proposed equipment will complement existing technical resources at the University including an Oligonucleotide Synthesizer, Cell Hybridoma Lab and Transmission Electron Microscope Facilities. It will serve active research programs in DNA and cDNA cloning, site directed mutagenesis, regulation of gene expression, and structural and functional characterization of proteins. In addition to the individual applications of each machine, a particular feature of these facilities is that they can be used effectively in conjunction. As examples, partial amino acid sequences will serve to define the possible sequences of an oligonucleotide hybridization probe, and these probes used in turn to isolate the genes encoding the initial protein. Conversely, from a DNA sequence one can unambiguously deduce the amino acid sequence. Peptides corresponding to parts of this sequence can be constructed, coupled to a carrier protein, and then used to generate antisera or monoclonal antibodies. These antibodies can be employed to characterize functionally the proteins by stimulating or blocking known functions. Finally, novel gene products may be identified using antibodies directed against peptides synthesized to correspond to open reading frames found by DNA sequence analysis. We envision these technologies complementing one another and the existing expertise in molecular biology, biochemistry, immunology, and tumor biology. They will allow a large number of investigators to address many fundamental questions of clinical and basic science.