Generating stable Chinese hamster ovary cell clones to produce a truncated SARS-CoV spike protein for vaccine development.
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.
Lin
Shih-Chang
SC
Institute of Biotechnology, Dept. of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan.
Leng
Chih-Hsiang
CH
Wu
Suh-Chin
SC
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
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
Vaccine. 2007 Jan 2;25(1):136-43
16919855
Biotechnol Bioeng. 2000 May 20;68(4):370-80
10745205
Nat Rev Microbiol. 2009 Mar;7(3):226-36
19198616
Biotechnol Prog. 2010 Jan-Feb;26(1):11-20
19847885
Biochem Biophys Res Commun. 2004 Nov 12;324(2):773-81
15474494
J Virol. 2008 Apr;82(7):3220-35
18199635
Nature. 2004 Apr 1;428(6982):561-4
15024391
BMC Microbiol. 2008 Nov 28;8:207
19038059
Vaccine. 2006 Apr 24;24(17):3624-31
16497416
J Biomed Sci. 2005 Oct;12(5):711-27
16132115
Nat Rev Microbiol. 2003 Dec;1(3):209-18
15035025
Biotechnol Bioeng. 1994 Mar 15;43(6):505-14
18615748
Nat Biotechnol. 2004 Nov;22(11):1393-8
15529164
Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9804-9
15210961
J Virol. 2004 Jul;78(13):7217-26
15194798
J Infect Dis. 2005 Feb 15;191(4):507-14
15655773
J Virol. 2006 Jun;80(12):5757-67
16731915
Science. 2004 Feb 13;303(5660):944-6
14963300
Biotechnol Bioeng. 1995 Jul 5;47(1):96-105
18623371
Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12995-3000
14569023
Virology. 2009 Oct 10;393(1):144-50
19683779
Biochem Biophys Res Commun. 2009 Jul 10;384(4):486-90
19422787
Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6641-6
15096611
Vaccine. 2007 May 16;25(20):4103-11
17428585
Vaccine. 2006 Feb 20;24(8):1132-41
16194584
Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2536-41
14983044
Science. 2003 May 30;300(5624):1399-404
12730501