EXPRESSION EFFICIENCY OF THE HUMAN THROMBOMODULIN-ENCODING GENE IN VARIOUS VECTOR AND HOST SYSTEMS

Expression systems were developed for evaluating recombinant human thr ombomodulin (TM) production in different host cell lines by investigat ing the performance of five mammalian expression vectors. The expressi on vectors were constructed so that they contain multiple monocistroni c gene cassettes which include a gene encoding a dominant selectable m arker, Hy(R) (hygromycin B phosphotransferase), under the regulation o f the thymidine kinase promoter, the target gene which encodes a trunc ated human re-TM under the regulation of various promoters, an amplifi able gene (Dhfr) encoding murine dihydrofolate reductase under the reg ulation of either the SV40 early or late promoter along with the SV40 enhancer and the SV40 ori. We tested the performance of the five expre ssion vectors in human embryonic kidney cells (HEK293), baby hamster k idney cells (BHK), human melanoma cells (CHL-1) and Dhfr(-) Chinese ha mster ovary cells (CHO/Dhfr(-)). We found that the efficiency of DNA u ptake, transient expression and stable expression of the different exp ression vectors were all cell-line dependent. However, the myeloprolif erative sarcoma virus (MPSV) LTR promoter consistently showed higher e xpression levels in all cell lines, particularly in HEK293 cells. Thes e results were confirmed by the distribution curves of the level of ex pression of individual clones. Furthermore, by amplifying Dhfr in tran sfected CHO/Dhfr(-) cells with 100 nM methotrexate, we achieved a 20-f old increase in re-TM production using the SV40 late promoter to contr ol murine Dhfr expression. Our data from DNA and mRNA analysis reveal that pMPSV-TM has a high transcription efficiency. Thus, we have devel oped a versatile expression system that is able to evaluate protein mo lecules rapidly by transient expression, or by generating stable clone s for large-quantity production and is amplifiable for ultimate protei n production.