Expression of Eta-1 in Escherichia coli and Production of Monoclonal Antibody

Lee, Sun Myung; Kim, Mee Kyung; Lim, Byung Uk; Cho, Hyeongjin; Kang, Jae Seung

Abstract

BACKGROUND: Early T-lymphocyte activation-1 (Eta-1) is a secreted phosphoprotein which regulates a variety of cells involved in the immune and nonimmune systems. It is unique in the sense that it regulates various immune functions, as well as acting as an extracellular matrix protein. The Eta-1 gene has been mapped to the same genetic locus as the Rickettsia resistance gene (Ric), and Eta-1 expression is a part of an early T-dependent response to Orientia tsutsugamushi infection in susceptible hosts. In an initial effort to study Eta-1's mechanism of protection against Orientia tsutsugamushi infection, we attempted to produce Eta-1 in E. coli and to produce monoclonal antibodies against recombinant Eta-1.
METHODS
Expression plasmids containing GST-Eta-1 were generated by cloning the polymerase chain reaction-amplified N-and C-terminal Eta-1 fragments into the cloning sites of pGEX-3X. The expressed protein was purified using a GST column and injected into BALB/c mice. Hybridoma clones reactive to Eta-1 were produced and analyzed with ELISA and Western blot.
RESULTS
Expression plasmids containing GST-Eta-1 were generated by cloning the polymerase chain reaction-amplified N-and C-terminal Eta-1 fragments into the cloning sites of pGEX-3X. N-and C-terminal fragments of Eta-1 were generated as bacterially expressed GST fusion proteins. However, the expression of full-length Eta-1 was very poor. We immunized BALB/c mice with purified Eta-1 N-terminal fragments. Their spleen cells were used for cell fusion. We obtained two hybridoma cell lines secreting antibodies against Eta-1, but not against GST.
Conclusions
We produced Eta-1 protein produced in E. coli. The expression of C-terminal Eta-1 fragments was poor, therefore it appeared that this part of Eta-1 was toxic to E. coli. We obtained monoclonal antibodies which were reactive in ELISA test and Western blot. These monoclonal antibodies could be useful in the analysis of the function of Eta-1 in the pathogenesis of tsutsugamushi disease as well as other diseases.