PRODUCTION OF RECOMBINANT EPIDERMAL GROWTH FACTOR RECEPTOR TYROSINE KINASE (EGFR-TK) DOMAIN IN AN INSECT CELL/BACULOVIRUS EXPRESSION SYSTEM

ABSTRACT The insect cell/baculovirus expression system was used to produce human EGFR-TK. Infection of Spodoptera frugiperda (Sf9) cells with a recombinant baculovirus (in which the EGFR-TK domain gene has been cloned downstream of the polyhedrin promoter) resulted in production of the authentic protein. We have successfully scaled-up production to the 30L scale using an airlift fermenter. The expressed protein recovered from the cells, had a molecular weight of ~66,000 daltons and was recognised by a polyclonal antibody to EGFR (whole receptor). The protein undergoes auto-phosphorylation and also phosphorylates synthetic substrates (angiotensin II, poly glu-tyr). EGFR-TK has been partially purified using anion exchange and gel permeation chromatography. When analysed on SDS PAGE following autophosphorylation, only a single band is detected indicating lack of other tyrosine kinases. Omission of sodium vanadate from the exogenous peptide assay (angiotensin II) showed no change in phosphorylation activity indicating the lack of phosphatases in the preparation.