title: 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. 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.
authors.affiliation: Institute of Biotechnology, Dept. of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Shih-Chang
authors.initials: SC
authors.lastname: Lin
authors.forename: Chih-Hsiang
authors.initials: CH
authors.lastname: Leng
authors.forename: Suh-Chin
authors.initials: SC
authors.lastname: Wu
mesh_terms: D000818:Animals; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics; D014765:Viral Vaccines / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus
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