Du Bois G C, Song S P, Kulikovskaya I, Virgilio L, Varnum J, Germann M W, Croce C M
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Protein Expr Purif. 1998 Mar;12(2):215-25. doi: 10.1006/prep.1997.0822.
The TCL-1 gene which is located on chromosome 14 plays a major role in human hematopoeitic malignancies and encodes a 14-kDa protein whose function has not been determined. The TCL-1 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 TCL-1 protein is similar in its primary structure to a protein encoded by the mature T cell proliferation gene (MTCP-1). The MTCP-1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T cell proliferative diseases. The TCL-1 and MTCP-1 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 TCL-1 and MTCP-1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, both purified TCL-1 and MTCP-1 proteins in forms with and without a six His tag sequence. 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 polymeric-based reverse-phase column. The MTCP-1 recombinant proteins display greater solubility, do not form disulfide linked dimers or oligomers, and elute at a lower isopropanol concentration than the corresponding TCL-1 proteins. The purified recombinant TCL-1 and MTCP-1 proteins have been characterized by N-terminal sequence analysis, time of flight mass spectrometry, and circular dichroism spectroscopy. Initial results have indicated that the MTCP-1 protein with the His tag removed is suitable for both NMR and X-ray crystallographic methods of structure determination.
位于14号染色体上的TCL-1基因在人类血液系统恶性肿瘤中起主要作用，它编码一种14 kDa的蛋白质，其功能尚未确定。TCL-1基因在pre-B细胞、未成熟胸腺细胞中表达，在活化T细胞中低水平表达，但在外周成熟B细胞和正常细胞中不表达。TCL-1蛋白的一级结构与成熟T细胞增殖基因（MTCP-1）编码的蛋白相似。MTCP-1基因位于X染色体上，已被证明与T细胞增殖性疾病中罕见的染色体易位有关。TCL-1和MTCP-1基因似乎是参与淋巴细胞增殖和T细胞恶性肿瘤的基因家族的成员。我们实验室已开展对TCL-1和MTCP-1蛋白的研究，以确定这些相关蛋白的结构和功能。在本报告中，我们使用细菌表达系统生产了带有和不带有六个组氨酸标签序列形式的纯化TCL-1和MTCP-1蛋白。重组蛋白通过镍-亚氨基二乙酸（Ni-NTA）树脂层析纯化，随后使用pH 7.9的缓冲系统和聚合物基反相柱进行反相快速蛋白质液相色谱（FPLC）。MTCP-1重组蛋白显示出更大的溶解度，不形成二硫键连接的二聚体或寡聚体，并且在比相应TCL-1蛋白更低的异丙醇浓度下洗脱。纯化的重组TCL-1和MTCP-1蛋白已通过N端序列分析、飞行时间质谱和圆二色光谱进行了表征。初步结果表明，去除组氨酸标签的MTCP-1蛋白适用于核磁共振（NMR）和X射线晶体学结构测定方法。
