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

Lin, Shih‐Chang; Leng, Chih‐Hsiang; Wu, Suh‐Chin

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. S(TR2) (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 S(TR2) expression. Our results indicate that the ∼115 kDa glycoform of S(TR2) protein was capable of increasing after gene amplification. The S(TR2) 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 S(TR2) on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010