Core--Protein Chemistry

人类TCL-1基因位于染色体14q32.1，在T细胞白血病和淋巴瘤中，通过染色体易位或倒位将TCL-1基因与T细胞受体的α / δ或β位点并置而激活。hTCL-1蛋白与Xq28染色体上的hTCL-1蛋白相同41%，相似61%，hTCL-1蛋白也参与T淋巴细胞增生性疾病。hTCL-1和htcp -1蛋白可能是参与淋巴细胞增殖和/或存活以及T细胞恶性肿瘤的癌基因家族的新成员。蛋白质化学核心实验室在本项目中的主要作用是制备和纯化毫克量的TCL-1和MTCP-1蛋白，这些蛋白进行功能分析和三维结构分析是必需的。hTCL-1和htmtcp -1蛋白在带6xHis和不带6xHis标记序列的大肠杆菌中均有表达，重组蛋白均以毫克为单位纯化。hTCL-1和htcp -1蛋白将使用真核表达系统生产，以毫克量纯化到均匀性，并将其与大肠杆菌生产的蛋白质进行比较。大肠杆菌系统也将用于hTCL-1突变体的制备。Core B将利用n端序列分析、电喷雾质谱（ES-MS）、等电聚焦（IEF）、凝胶过滤层析和聚丙烯酰胺凝胶电泳（PAGE）等技术，对该实验室生产的所有重组TCL-1和MTCP-1蛋白的特性进行研究。蛋白质化学核心将利用[32P]标记和荧光探针的光结合、化学或酶方法的碎片化、HPLC分离和肽测序，绘制TCL-1和MTCP-1蛋白的推定核苷酸结合位点和暴露的疏水位点。Core B将开展研究，确定野生型和突变型TCL-1和MTCP-1与相互作用蛋白（如EWS和TAF/II68）的结合，并利用生物传感器技术进一步研究确定TCL-1和MTCP-1的新分子伙伴。Core还将制备TCL-1和MTCP-1的单克隆和多克隆抗体、突变形式、肽和相互作用蛋白。Core B将采用固相合成方法制备所有通过分子建模设计的TCL-1和MTCP-1的肽类似物，以作为其蛋白质功能的潜在抑制剂。

The human TCL-1 gene maps at chromosome 14q32.1 and is activated in T cell leukemias and lymphomas by either chromosome translocations or inversions that juxtapose the TCL-1 gene to the alpha/delta or beta locus of the T cell receptor. The hTCL-1 protein is 41% identical and 61% similar to the hMTCP-1 protein on chromosome Xq28 which is also involved in T cell lymphoproliferative diseases. The hTCL-1 and hMTCP-1 proteins may be new members of an oncogene family involved in lymphoid proliferation and/or survival and in T cell malignancies. The major role of the Protein Chemistry Core Laboratory in this project is to produce and purify the TCL-1 and MTCP-1 proteins in milligram amounts which are necessary to conduct functional analysis and three- dimensional structural analysis of these proteins. The hTCL-1 and hMTCP-1 proteins have been expressed in C. coli both with and without 6xHis tag sequence, and the recombinant proteins have been purified to homogeneity in milligram amounts. The hTCL-1 and hMTCP-1 proteins will be produced using a eukaryotic expression system, purified to homogeneity in milligram amounts, and their properties compared to the E. coli produced proteins. The E. coli system will also be used for the preparation of mutant forms of hTCL-1. Core B will conduct studies to characterize the properties of all recombinant TCL-1 and MTCP-1 proteins produced in this laboratory using the techniques of N-terminal sequence analysis, electrospray mass spectrometry (ES-MS), isoelectric focusing (IEF), gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). The Protein Chemistry Core will map putative nucleotide binding sites and the exposed hydrophobic sites of both TCL-1 and MTCP-1 proteins using photoincorporation of [32P]-labeled and fluorescent probes, fragmentation by chemical or enzymatic methods, separation by HPLC, and peptide sequencing. Core B will conduct studies to determine the binding of wild-type and mutant forms of TCL-1 and MTCP-1 to interacting proteins such as EWS and TAF/II68 and also further studies to identify new molecular partners of TCL-1 and MTCP-1 using the biosensor technology. The Core will also prepare monoclonal and polyclonal antibodies to TCL-1 and MTCP-1, mutant forms, peptides, and interacting proteins. Core B will prepare by solid phase synthesis all peptide analogs of TCL-1 and MTCP-1 designed by molecular modeling to act a potential inhibitors of their protein function.