In mammals, an adequate supply of thyroid hormones is essential for normal growth and neurological development. The biosynthesis of thyroid hormones involves an iodinated precursor protein, thyroglobulin, which may be considered an extreme example of a pro-hormone. Thyroglobulin is a dimeric glycoprotein of relative molecular mass (Mr) 660,000 (660K), which is secreted by the thyrocyte and stored in the lumen of the thyroid follicle. The hormonogenic reaction is extracellular, and involves iodination of tyrosyl residues of thyroglobulin and the intramolecular coupling of a subset of these into thyroxine (T4) and triiodothyronine (T3), which remain part of the polypeptide chain. Secretion of hormones results from the endocytosis of thyroglobulin followed by its complete hydrolysis in lysosomes. Considering that the maximum yield of hormones is approximately 6-8 per 660K protein, the whole process is apparently wasteful. However, the efficiency of thyroglobulin as a thyroid hormone precursor is extremely high when the supply of iodine is short; in such conditions, almost all the iodine incorporated is found in iodothyronine. Hence it is suggested that the thyroglobulin structure has evolved to allow for the preferential and efficient iodination and coupling of the hormonogenic tyrosines. Here we report the complete primary structure of bovine thyroglobulin, derived from the sequence of its 8,431-base-pair complementary DNA. The 2,769-amino-acid sequence is characterized by a pattern of imperfect repeats derived from three cysteine-rich motifs. Four hormonogenic tyrosines have been precisely localized near the amino and carboxyl ends of the protein.

Background: The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells. Methods: As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction. Results: COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5–10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1. Conclusions: These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.

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Varicella-zoster virus (VZV) gene 63 encodes a protein (IE63) with a predicted molecular mass of 30.5 kIDa which has amino acid similarities to the immediate-early (IE) protein 22 (ICP22) of herpes simplex virus type 1. ICP22 is a polypeptide synthesized in herpes simplex virus type 1-infected cells, and as is the case for its VZV counterpart, its regulatory functions are unknown. On the basis of the VZV DNA sequence, it has been shown that IE63 exhibits hydrophilic and acidic properties, suggesting that this protein could play a regulatory role during the infectious cycle. We report in this article cotransfection experiments which demonstrate that the VZV gene 63 protein strongly represses, in a dose-dependent manner, the expression of VZV gene 62. On the other hand, transient expression of the VZV gene 63 protein can promote activation of the thymidine kinase gene but cannot affect the expression of the genes encoding glycoproteins I and II. The results of transient expression experiments strongly suggest that the VZV gene 63 protein could play a pivotal role in the repression of IE gene expression as well as in the activation of early gene expression.