Purification and characterization of recombinant forms of murine Tcl1 proteins

The TCL1 gene, which is located on chromosome 14, plays a major role in hum an hematopoietic malignancies and encodes a 14-kDa protein whose function h as 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 Tell protein is similar in its prim ary 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 t o be involved in rare chromosomal translocations in T-cell proliferative di seases. The murine TCL1 gene resides on mouse chromosome 12 and is homologo us to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino aci d residues and shares 50% sequence identity with human Tcl1, while the huma n and mouse Mtcp1 are nearly identical, with conservative differences in on ly 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 la boratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determi ne the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purifie d murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant pr oteins were purified by chromatography on a Ni-NTA resin followed by revers e-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-ph ase column. The murine Tell recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine T ell C86A protein has increased solubility and does not form higher order ol igomers, The purified recombinant murine proteins were characterized by N-t erminal sequence analysis, mass spectrometry, and circular dichroism spectr oscopy. Initial results indicate that the mutant murine Tell C86A protein i s suitable for both NMR and X-ray crystallographic methods of structure det ermination. (C) 2000 Academic Press.