Gene Expression Of Epidermal Growth Factor Receptor (EGFR) And Fms-like Tyrosine Kinase (FLT3) In Yeast Pichia Pastoris

Epidermal growth factor receptor (EGFR) and Fms-like tyrosine kinase (FLT3 ) are important transmembrane receptors with signal-transducing tyrosine kinase activity. The EGFR signal plays a fundamental role in the development and growth of many types of human tumour cells. FLT3 which abnormally expresses has been identified in mulitiple myeloid malignancies in recent years. Exploring the pathogenesis at the molecular level can accelerate the diagnosis and treatment in cancers.Pichia pastoris is an excellent expression system of the heterologous proteins. Due to the high expression, high stability, high secretion, easy purification and low cost etc., Pichia pastoris expression system is chosen to expresse EGFR and FLT3. The fragments of EGFR and FLT3 were subcloned into pPICZαA vector and sequencing, respectively. The pPICZαA-EGFR and pPICZαA-FLT3 were separately linearized by SacI and BstX I, transformed into P. pastoris X33 by electroporation. Positive clones and Transformants with multicopy were selected by zeocin. To identify the best strain for expression of our protein, in additon to methanol, other culture conditions such as time of induction, potassium phosphate buffer (pH6.0), were examined as well. SDS-PAGE analysis showed that the supernatant of the induced P. pastoris culture contained protein. The molecular weight of FLT3 was about 58kDa, and EGFR was about 68kDa. The maximal expression of EGFR was ' obtained at 28℃after induction for 84h with the 1.5% of methanol, and FLT3 was obtained at 28℃after induction for 96h with the 1.5% of methanol. The recombinant protein was purified by ammonium sulfate precipitation, followd by Ni-NTA agarose Column.