Purification and characterization of recombinant forms of TCL-1 and MTCP-1 proteins.
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.
Du Bois
G C
GC
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Song
S P
SP
Kulikovskaya
I
I
Virgilio
L
L
Varnum
J
J
Germann
M W
MW
Croce
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007106:Immune Sera; C082870:MTCP1 protein, human; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C490393:Tcl1 protein, mouse