Expression and Purification of Receptor Tyrosine Kinase PDGFRβ

Abstract:

A fusion expression vector pPIC3.5K-PDGFRβ was constructed to express recombinant receptor tyrosine kinase PDGFRβ and the right Pichia. pastoris transformants were screened on his-deficient plates and YPD-G418 plates by turns after electroporation of strain GS115, a high yield strain named M3 was screened. The strain M3 was cultured in a 5 L fermentor and His-GFP-PDGFRβ fusion protein was purified by Ni2+ chelating affinity chromatography. One distinct peak was obtained after elution with 250 mmol/L imidazole. Fusion protein was proved to be 90.08 kD by western blotting, and have tyrosine kinase activity by ELISA. Results showed that the receptor tyrosine kinase PDGFRβ was successfully expressed in P. pastoris and could be used as a target for small molecule selective inhibitors screening.