Purification and characterization of recombinant forms of murine Tcl1 proteins.-猫眼课题宝

鼠 Tcl1 蛋白重组形式的纯化和表征。

DOI：

10.1006/prep.1999.1186

发表时间：

2000

期刊：

Protein expression and purification

影响因子：

1.6

通讯作者：

中科院分区：

生物学4区

文献类型：

作者：

G. C. Du Bois;Sherry P. Song;Irina Kulikovskaya;Jay L. Rothstein;Markus W. Germann;C. M. Croce

TCL1基因位于14号染色体上，在人类血液系统恶性肿瘤中起重要作用，编码一个14 kDa的蛋白，其功能尚未确定。该基因在前B细胞、未成熟胸腺细胞和低水平激活的T细胞中表达，但在外周成熟B细胞和正常细胞中不表达。TCL1蛋白在一级结构上与成熟T细胞增殖基因(MTCP1)编码的蛋白相似。MTCP1基因位于X染色体上，已被证明与T细胞增生性疾病中罕见的染色体易位有关。小鼠TCL1基因位于小鼠12号染色体上，与人类TCL1和MTCP1基因同源。小鼠TCL1蛋白有116个氨基酸残基，与人TCL1有50%的序列同源性，而人和小鼠的Mtcp1几乎相同，保守的差异只有6个残基。TCL1和MTCP1基因似乎是与淋巴增殖和T细胞恶性肿瘤有关的基因家族的成员。我们实验室对TCL1和Mtcp1蛋白进行了研究，以确定这些相关蛋白的结构和功能。在本报告中，我们利用细菌表达系统，获得了纯化的小鼠TCL1蛋白和突变形式的小鼠TCL1蛋白，该蛋白在第85位含有半胱氨酸到丙氨酸的突变。重组蛋白经Ni-NTA树脂层析、反相FPLC、pH 7.9的缓冲体系和聚合物反相柱纯化。小鼠TCL1重组蛋白的溶解度有限，并形成二硫键连接的二聚体和低聚物，而突变的小鼠TCL1 C86A蛋白的溶解度增加，不形成更高阶的低聚物。用N-末端序列分析、质谱学和圆二色谱对纯化的重组鼠蛋白进行了表征。初步结果表明，突变的小鼠TCL1C86A蛋白适用于核磁共振和X射线结晶学结构测定方法。

The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination.