title: Purification and characterization of recombinant forms of murine Tcl1 proteins.
abstract: 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.
authors.affiliation: Department of Microbiology, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107, USA. G_Dubois@iac.jci.tju.edu
authors.forename: G C
authors.initials: GC
authors.lastname: Du Bois
authors.forename: S P
authors.initials: SP
authors.lastname: Song
authors.forename: I
authors.initials: I
authors.lastname: Kulikovskaya
authors.forename: J L
authors.initials: JL
authors.lastname: Rothstein
authors.forename: M W
authors.initials: MW
authors.lastname: Germann
authors.forename: C M
authors.initials: CM
authors.lastname: Croce
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D000906:Antibodies; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C090851:TCL1A protein, human; C490393:Tcl1 protein, mouse