Recombinant human parathyroid hormone synthesized in Escherichia coli. Purification and characterization.

Rabbani, Shafaat A.; Yasuda, Toshiyuki; Bennett, H. P. J.; Sung, Wing L.; Zahab, Diana M.; Tam, Carolyn; Goltzman, David; Hendy, Geoffrey N.

doi:10.1016/s0021-9258(19)57301-x

Cited by 43 publications

(11 citation statements)

References 22 publications

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“…Progressive truncation of C-terminal residues from PTH(1-34) caused a marked loss in the potency on wild-type PTHR1 and other PTHR1 variants with intact ECDs (Table 1, Supporting Figure 5). Addition of a triglycine appendage at the N-terminus of PTH(1-14) caused a reduction in potency, relative to PTH(1-14) with a free N-terminal amine (Supporting Figure 6), in line with precedent 31 . Conjugates in which G3-PTH(1-14) was ligated to the VHH C-terminus using sortase were completely inactive, emphasizing the importance of a free N-terminus for PTH (Supporting Figure 6).…”

Section: Peptide Ligands Of Pthr1 and Conjugation To Antibody Fragments To Test Whether Deliverysupporting

confidence: 60%

Improved GPCR Ligands Based on Genetically Impossible Nanobody-Peptide Fusions

Cheloha

Fischer²,

Woodham³

et al. 2019

Preprint

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Section: Peptide Ligands Of Pthr1 and Conjugation To Antibody Fragments To Test Whether Deliverysupporting

confidence: 60%

Improved GPCR Ligands Based on Genetically Impossible Nanobody-Peptide Fusions

Cheloha

Fischer²,

Woodham³

et al. 2019

Preprint

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“…Model 380B. Enzymes were purchased from Bethesda Research Laboratory [T4 DNA ligase, calf intestinal alkaline phosphatase (CIAP), and restriction endonucleases EcoRI, BamHl, and Ps/I] and New England Biolabs (T4 Rabbani et al (1988b) and Sung et al (1986a,b) for description of the oligonucleotides]. Figure 1A outlines the construction scheme of plasmid pPTH-(3-84).…”

Section: Methodsmentioning

confidence: 99%

“…Expression in E. coli. E. coli transformants bearing either pPTH-(l-84), pPTH-(3-84), or pPTH-(-6-+84) were grown in Luria broth containing ampicillin, and hPTH gene tran-scription was induced with 1 mM IPTG as described previously (Rabbani et al, 1988b). Cells were harvested for radioimmunoassay before and after addition of chloramphenicol to inhibit protein synthesis, as described (Rabbani et al, 1988b).…”

Section: Methodsmentioning

confidence: 99%

“…Messenger RNA Analysis. E. coli harboring either the pPTH-(l-84), pPTH-(3-84) or pPTH-(-6-H-84) plasmids were grown in Luria broth containing ampicillin, and hPTH gene transcription was induced with 1 mM IPTG as described previously (Rabbani et al, 1988b). After 7 h at 37 °C, 1-mL aliquots of cells were harvested, and RNA was isolated as described (Ivanov et al, 1987), treated with 10 /tg/mL DNase I (RNase free), deproteinized by phenol extraction, and precipitated with ethanol.…”

Section: Methodsmentioning

confidence: 99%

“…Peptide Extraction Procedure. Cells were homogenized in acetone and n-hexane, and the dried residue was extracted in a mixture of 1 M HC1, 5% (v/v) formic acid, 1% (w/v) NaCl, and 1% (v/v) trifluoroacetic acid (CF3COOH), as described previously (Rabbani et al, 1988b;Bennett et al, 1981).…”

Section: Methodsmentioning

confidence: 99%

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Synthesis and characterization of extended and deleted recombinant analogs of parathyroid hormone-(1-84): correlation of peptide structure with function

Rabbani¹,

Kaiser²,

Henderson³

et al. 1990

Biochemistry

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Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

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Gensynthese

Engels

Uhlmann²

1989

Angewandte Chemie

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Oligonucleotid-Synthese, bis vor einigen Jahren noch eher ein exotisches Betatigungsfeld weniger Experten, ist mittlerweile integraler Bestandteil des Arsenals molekularbiologischer Techniken. Gerade im letzten Jahrzehnt hat sich auf dem Gebiet der DNA-Synthese eine unglaublich rasante Entwicklung abgespielt, welche zur Automatisierung der Synthesen fiihrte und schlieBlich in der Herstellung von Genen mit einer Lange von iiber 1000 Basenpaaren gipfelte. MaBgeschneiderte synthetische Gene ermoglichen heute auch die gentechnische Herstellung veranderter und sogar vollig neuartiger Proteine (,,de-novo-Protein-Design"). Im Zusammenspiel mit den modernen Methoden der Isolierung, Sequenzierung und Expression von Genen trug die Gensynthese in den letzten Jahren wesentlich zum enormen Fortschritt in der Gentechnologie bei. Synthesemethoden fur OligonucleotideDie Methoden zur Synthese definierter Oligonucleotide unterscheiden sich im wesentlichen durch die Art, wie die Phosphorsaureesterbindung erhalten wird. Je nach Art Angew Chem. 101 (1989) 733-752 C VCH VerlugsgeseIlsc.hafi mbH, 0-6940 Weinhrim, I989

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Recombinant human parathyroid hormone synthesized in Escherichia coli. Purification and characterization.

Cited by 43 publications

References 22 publications

Improved GPCR Ligands Based on Genetically Impossible Nanobody-Peptide Fusions

Improved GPCR Ligands Based on Genetically Impossible Nanobody-Peptide Fusions

Synthesis and characterization of extended and deleted recombinant analogs of parathyroid hormone-(1-84): correlation of peptide structure with function

Gensynthese

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