Core--Protein Chemistry

人TCL-1基因定位于染色体14q32.1，并且在T细胞白血病和淋巴瘤中通过染色体易位或倒位被激活，所述染色体易位或倒位将TCL-1基因与T细胞受体的α/δ或β基因座并置。hTCL-1蛋白与染色体Xq28上的hMTCP-1蛋白有41%的相同性和61%的相似性，其也参与T细胞淋巴增生性疾病。hTCL-1和hMTCP-1蛋白可能是参与淋巴增殖和/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该项目中的主要作用是生产和纯化毫克量的TCL-1和MTCP-1蛋白质，这是对这些蛋白质进行功能分析和三维结构分析所必需的。hTCL-1和hMTCP-1蛋白已在C.大肠杆菌中表达的重组蛋白经纯化后均达到毫克级纯度。hTCL-1和hMTCP-1蛋白将使用真核表达系统产生，纯化至毫克量的均一性，并且将它们的性质与E.大肠杆菌产生蛋白质。急诊coli系统也将用于制备hTCL-1的突变体形式。核心B将采用N-末端序列分析、电喷雾质谱（ES-MS）、等电聚焦（IEF）、凝胶过滤色谱和聚丙烯酰胺凝胶电泳（PAGE）技术进行研究，以表征本实验室生产的所有重组TCL-1和MTCP-1蛋白的特性。蛋白质化学核心将使用[32P]标记和荧光探针的光掺入、化学或酶促方法的片段化、HPLC分离和肽测序，绘制TCL-1和MTCP-1蛋白质的推定核苷酸结合位点和暴露的疏水位点。核心B将进行研究，以确定TCL-1和MTCP-1的野生型和突变型与相互作用蛋白（如EWS和TAF/II 68）的结合，并进一步研究，以使用生物传感器技术鉴定TCL-1和MTCP-1的新分子伴侣。核心还将制备针对TCL-1和MTCP-1、突变体形式、肽和相互作用蛋白的单克隆和多克隆抗体。核心B将通过固相合成制备TCL-1和MTCP-1的所有肽类似物，这些肽类似物通过分子建模设计以充当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.