Reversed-phase high-performance liquid chromatography method for the determination of prolactin in bacterial extracts and in its purified form.

Reversed-phase high-performance liquid chromatography methodology for the determination of human prolactin (hPRL) in bacterial periplasmic space or in purified preparations has been developed. The technique, based on the high hydrophobicity of the hPRL molecule, allows its separation from the bulk of bacterial proteins. The precision for periplasmic shock fluid analysis was characterized by relative standard variations of 3-7% for intra-day and of 3-25% for inter-day determinations. Accuracy, evaluated by recovery tests, was of the order of 90%, a calibration curve being constructed with the use of a lyophilized osmotic shock fluid extract, which provided a stable, readily prepared internal reference. Sensitivity was of the order of 0.5 microg of hPRL. The methodology developed also provided a tool for comparing the hydrophobicity of glycosylated and non-glycosylated prolactin molecules obtained from several different species and of different preparations of native or biosynthetic human prolactin.

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.