Cloning, sequencing, and expression of mouse c-ets-1 cDNA in baculovirus expression system.

Abstract

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.