Cloning, sequencing, and expression of mouse c-ets-1 cDNA in baculovirus expression system.
The protooncogene c-ets-1 is preferentially expressed in lymphoid cells. The protein product of this gene has been found to be a phosphorylated nuclear protein. When lymphocytes are stimulated with calcium ionophore, hyperphosphorylation of c-ets-1 occurs. In order to study the biological and biochemical functions of the c-ets-1 protein in detail, it is important to prepare adequate quantity of the c-ets-1 protein. To this end, we have cloned, sequenced, and expressed mouse c-ets-1 cDNA in baculovirus expression vector. Sequence analysis indicated that mouse c-ets-1 cDNA codes for a 50/51-kd protein. Since the mouse c-ets-1 protein in mouse lymphocytes is a 60/62-kd protein, the result obtained indicated that the c-ets-1 protein undergoes posttranslational modification by phosphorylation. When the c-ets-1 cDNA was expressed in the baculovirus expression vector, insect cells infected with a recombinant virus synthesizes a protein of the same size but with 50-100 times more of the c-ets-1 protein than that of mouse lymphocytes. The Staphylococcus aureus V8 protease mapping analysis of mouse c-ets-1 proteins synthesized in mouse and insect cells showed that they are identical. Thus, the c-ets-1 protein synthesized in insect cells will allow us to purify and study the functions of the c-ets-1 protein in detail.
Chen
J H
JH
Department of Pediatrics, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta / Q000166:cytology; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D050783:Proto-Oncogene Proteins c-ets; D015183:Restriction Mapping; D014157:Transcription Factors
D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D004247:DNA