We present a new method for rapid purification to near homogeneity of sequence specific DNA binding proteins based on magnetic separation. The method is described for the purification of the yeast transcription factor tau. DNA affinity Dynabeads (monodisperse superparamagnetic particles) specifically bind the protein in the presence of competitor DNA. By magnetic separation, wash and elution, highly enriched transcription factor preparations are obtained within minutes. In less than an hour with three cycles of adsorption, nearly homogeneous factor tau was obtained. The factor preparation contained mainly two polypeptides of 100 and 140 kDa and was fully active in transcription and DNA binding assays. This procedure should work for any high-affinity sequence-specific DNA binding protein with only minor modifications.

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Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the 0-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for 0-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal 0-glycosylation of hPTH with upto seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.Parathyroid hormone is the principle regulator of calcium homeostasis in human and has been advanced as an anabolic drug against post-menopausal osteoporosis [l, 21. The hormone, which is produced in the mammalian parathyroid glands, is synthesized as an 11 5-amino-acid precursor that is processed to the mature hormone of 84 amino acids [3].The physiological action of human parathyroid hormone (hPTH) is probably to generate a positive calcium balance and bone formation through its direct effects on the kidney and skeleton and through indirect effects via renal hydroxylation of 25-hydroxy-vitamin D3 to the active 1,25-dihydroxyvitamin D3. Ahhreviations. hPTH, human parathyroid hormone; hPTH-glycl , recombinant human parathyroid hormone containing one 0-glycosylation site; hPTH-glyc2, recombinant human parathyroid hormone containing two 0-glycosylation sites; MFa, yeast mating factor alpha; Pa-n, theoretical fragments after digestion with protease from Stuphylococcus rrureus, where n is the number given to the fragments starting from the N-terminus; T-n, theoretical fragments after digestion with trypsin where n is the number given to the fragments starting from the N-terminus.Due to shortage of supply, only the N-terminal 1-34 amino acid fragment of hPTH has been used in most experiments and clinical trials. One rational was that this peptide fragment, which is not a natural metabolic product, does, however, have biological activity [4].We show that cloned hPTH cDNA [5] expressed in Saccharomyces cerevisiae under the control of the o! mating factor (MFa) promoter [6] results in secretion into the medium of both the intact hormone and two 0-glycosylated forms...

When yeast cell extracts that faithfully transcribe class III genes are provided with different electrolyte ions, the pattern of transcripts changes. A transcription unit in pBR322, silent with 0.1M potassium chloride, becomes active in the presence of 0.1M potassium acetate. This pseudogene depends on transcription factors B and C and RNA polymerase III like a tRNA gene. The transcribed region contains the only sequence in pBR322 homologous to the modified B block consensus sequence GTTCRDNNC found in normal tRNA genes. The presence of a block A sequence is less evident. When a block A deleted tRNA(GLU) gene was constructed, it behaved similarly: poorly transcribed with 0.1M potassium chloride, well transcribed with 0.1M potassium acetate. In fact, the deletion of the A block promoter element from the tRNA(GLU) gene did not dramatically lower its transcription when tested with potassium acetate, while it had a strong negative effect when tested with potassium chloride. Consequently the requirement for this promoter element is not constant but is a function of the electrolyte composition.

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