Purification and immunodetection of the complete recombinant HER-2[neu] receptor produced in yeast

Abstract

For the first time, the full length recombinant HER-2[neu] receptor has been produced in a yeast (Arxula adeninivorans). It is one of the most studied membrane receptors in oncology and is involved in aggressive tumor formation. A yeast integration rDNA cassette containing the human gene coding for the HER-2[neu] protein was constructed and a screening procedure was performed to select the most productive transformant. Different detergents were tested for efficient solubilization of the membrane bound protein, with CHAPS giving the best results. To increase the yield of the recombinant protein from HER-2[neu] producing A. adeninivorans, optimal culture parameters were established for cultivation in bioreactor. The recombinant protein was subsequently assayed using ELISA and SPR immunoassays systems with antibodies raised against two different epitopes of the human receptor. In both cases, elution fractions containing the recombinant HER-2[neu] receptor successfully reacted with the immunoassays with limits of quantification below 100ngml(-1). These results demonstrate that the full length recombinant HER-2[neu] reported here has the potential to be a new standard for the detection of HER-2 type cancer.

Keywords: A. adeninivorans; Breast cancer; HER-2[neu]; Immunoassay; Protein solubilization; Surface Plasmon Resonance.