Synthesis of human parathyroid-hormone-related protein(1-141) in Saccharomyces cerevisiae. A correct amino-terminal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach.
Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein. A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid-hormone-related protein (hPTHrP) to mediate activation of adenylate cyclase. We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short-lived hPTHrP(1-141) as a carboxyl extension to ubiquitin in yeast. A hybrid gene containing the hPTHrP(1-141) cDNA coding region fused in-frame to the 3' end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter. The recombinant protein was purified to homogeneity and finally characterized by N-terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin-specific yeast endoprotease to generate authentic hPTHrP(1-141). hPTHrP(1-141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1-84) and [Tyr34]hPTHrP(1-34)amide. Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1-141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain-specific activities and biological roles.
Institute of Medical Biochemistry, University of Oslo, Norway.
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E
Rian
R
R
Jemtland
O K
OK
Olstad
J O
JO
Gordeladze
K M
KM
Gautvik
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004274:DNA, Recombinant; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000235:genetics*
D004274:DNA, Recombinant; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C066078:parathyroid hormone-related protein (1-141); D000262:Adenylyl Cyclases