Generating stable chinese hamster ovary cell clones to produce a truncated SARS-CoV spike protein for vaccine development

Abstract

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. STR2 (an 88 kDa truncated SARS-CoV TW1 S protein carrying the S fragments S-74-253, S-294-739, and S-1129-1255) is capable of expressing a major form of glycoprotein as endo H-sensitive (~115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr-cells with the amplifiable vectors ISID (IRES-driven dhfr) and ISIZ (SV40-driven dhfr) to select stepwise MTX, and observed enhanced ~115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES-driven dhfr promoter generates greater gene amplification, which in turn enhances STR2 expression. Our results indicate that the ~115 kDa glycoform of STR2 protein was capable of increasing after gene amplification. The STR2 glycoform did not change between suspension and serum-free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous STR2 on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 © 2010 American Institute of Chemical Engineers (AIChE).