High-level expression of human thyroid-stimulating hormone in Chinese hamster ovary cells by co-transfection of dicistronic expression vectors followed by a dual-marker amplification strategy.
The utilization of dicistronic mRNA expression vectors, containing the gene of interest upstream of an amplifiable marker gene, has shown success in rapidly, efficiently and reproducibly obtaining stable cell lines that express high levels of the protein of interest. For this reason, human thyroid-stimulating hormone (hTSH), a heterodimeric glycoprotein composed of non-covalently linked alpha- and beta-subunits, was expressed in Chinese hamster ovary (CHO) cells using a system based on dicistronic expression vectors. These contained the genes of interest and the amplifiable gene markers dihydrofolate reductase (DHFR) and adenosine deaminase (ADA), separated by an internal ribosome entry site isolated from the encephalomyocarditis virus. After the cells (CHO-DHFR-) had been co-transfected with the expression vectors and submitted to gene amplification in culture medium containing stepwise increments of methotrexate, it was possible to isolate clones that presented a secretion level of up to 7.2+/-1.3 microg/10(6) cells per day, the highest ever reported for the expression of this glycoprotein hormone. A second treatment, involving the utilization of deoxycoformycin, directed to amplify the ADA marker gene, provided a clone with an additional 2-3-fold increase in hTSH secretion, reaching a secretion level of 17.8+/-7.6 microg/10(6) cells per day. Cell culture and hTSH production in a hollow-fibre bioreactor were set up in order to carry out a preliminary physico-chemical, immunological and biological characterization of this hormone in comparison with pituitary-extracted hTSH (from the National Institute of Diabetes and Digestive and Kidney Diseases) and the only recombinant hTSH now available (Thyrogen). The availability of recombinant hTSH is very important in the diagnosis and therapy of thyroid carcinoma, via stimulation of radioiodine uptake.
Peroni
Cibele N
CN
Biotechnology Department, National Nuclear Energy Commission (IPEN-CNEN), Travessa R-400, Cidade Universitária, 05508-900, São Paulo, SP, Brazil.
Soares
Carlos R J
CR
Gimbo
Elizabeth
E
Morganti
Ligia
L
Ribela
Maria Teresa C P
MT
Bartolini
Paolo
P
D000243:Adenosine Deaminase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D006224:Cricetinae; D005260:Female; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015870:Gene Expression; D005796:Genes; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015649:Pentostatin / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013972:Thyrotropin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D005493:Folic Acid Antagonists; D005819:Genetic Markers; D011994:Recombinant Proteins; D015649:Pentostatin; D013972:Thyrotropin; D013762:Tetrahydrofolate Dehydrogenase; D000243:Adenosine Deaminase; D008727:Methotrexate