Protein Expression of CHO cells during Dihydrofolate Reductase Mediated Gene Amplification

1. Protein Expression of CHO cells during Dihydrofolate Reductase Mediated Gene Amplification 2006년 6월 12일 약학과 석사2학년김 윤 선

2. dhfr- CHO and the amplifiable selectable marker, DHFR DHFR Gene of interest • most popular mammalian expression system within the pharmaceutical industry – rapid production of clinically useful amounts of product • DHFR gene : coding for DHFR enzyme, which catalyses the conversion of dihydrofolate to tetrahydrofolate ( need to DNA synthesis) • The metothrexate (MTX) binds to the DHFR enzyme and inhibits the function. But DHFR-deficient CHO cells that have taken up an expression vector containing DHFR gene can develop resistance to MTX by amplifying the DHFR gene • Since the amplification unit is much larger ( 100-3000kb) than the size of DHFR gene, the specific gene of interest, which is co-linked with the DHFR gene in the same expression vector is coamplified

3. Expression Vectors for Human-mouse Chimeric Antibodies Journal of Biochemistry and Molecular Biology Vol. 38, No. 4, July 2005, pp. 414-419

4. SphI SphI SV40 promoter DHFR pSV-dhfr ampR SV40 Digest with SphI SphI SV40 promoter DHFR hCMV promoter MCS BGH polyA pYR-SV2-rdhfr ampR SV40 Digest with BamHI EcoRI, ligation SV40 promoter SV40 promoter ampR DHFR DHFR BGH polyA pYR-GSEVH pYR-GCEVH SV40 SV40 ampR Digest with XhoI , insertion Cg Cg MCS hCMV hCMV BGH polyA XhoI NotI BamHI NotI BamHI Cg Construction of the expression vector Heavy chain

5. BGH polyA ampR SV40 promotor ampR DHFR BGH polyA SV40 ori pYR-GCEVL pYR-GCEVH Neo SV40 Cl or Ck SV40 hCMV Cg hCMV BamHI NotI NotI BamHI Heavy chain Light chain

6. Antibody production by transfected cells • cell : dhfr- Chinese hamster ovary cell (CHO) • transfection : LipofectAMINE • selection : DHFR+ Neo+ clones 1. culture the cells in glycine, hypoxanthine, thymidine (GHT) free media 2. G418 • selected clones were subcloned in 96-well microtiter plate • The culture supernatant was tested for assembled antibody production by ELISA • The transfected CHO clones were subjected to amplification - stepwise increments in MTX level

7. Western blot analysis of Ab product Average productivity of Ab 3*10-8M MTX – 30mg/ml 1*10-6M MTX – 100mg/ml

8. Expression of chimeric antibody in mammalian cells using dicistronic expression vector Biotechnology Letters (2005) 27: 1713--1717

9. Schematic diagram of pDHL and pDHL-DC

10. The chimeric antibody production. (□ pDHL-DC ■ pDHL).