ABSTRACT
Through the use of oligonucleotide-directed mutagenesis we have generated variants of a recombinant human parathyroid (PTH) hormone-(1-34)-homoserine (RPTH) in which a positively charged residue (Arg or Lys), a negatively charged residue (Glu), or a neutral residue (Gly) has been substituted at every position throughout the peptide. These 106 PTH analogs have been tested for their ability to stimulate cAMP production in the rat osteosarcoma cell line, UMR106. Analysis of these peptides led to the construction of several analogs containing multiple substitutions at sites of potential structural importance. Several of these analogs were shown to have 3-5-fold enhanced activity and receptor affinity. Circular dichroism (CD) and lipid binding studies were then performed on these analogs. Circular dichroism demonstrates enhanced helical content in the presence of lipid vesicles, particularly anionic lipid. The [Arg15,19,22,Lys29]RPTH (+6RPTH) analog requires higher concentrations of trifluoroethanol to attain enhanced helicity. The intrinsic tryptophan fluorescence of the peptides are blue shifted more in the presence of the anionic lipid dimyristoyl phosphatidylglycerol (DMPG) than with the zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC). Effects of the peptides on the phase transition behavior of DMPC shows that +6RPTH has less effect on the lipid than does RPTH. This difference in lipid interaction is also exhibited with isothermal titration calorimetry, in which RPTH reacts exothermally with DMPG, while +6RPTH shows little or no heat change. The pH dependence of binding of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-trisulfonic acid, also shows a difference in exposure of hydrophobic sites between RPTH and +6RPTH. The +6RPTH has about a 5-fold greater affinity for receptor binding. We suggest that this enhanced activity is a consequence of the altered lipid interaction of +6RPTH, combined with increased conformational flexibility, particularly in the carboxyl-terminal region of the molecule.
Subject(s)
Parathyroid Hormone/chemistry , Parathyroid Hormone/pharmacology , Phospholipids/metabolism , Protein Conformation , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Calorimetry, Differential Scanning , Cell Line , Chromatography, High Pressure Liquid , Circular Dichroism , Cloning, Molecular , Cyclic AMP/metabolism , Humans , Molecular Sequence Data , Osteosarcoma , Parathyroid Hormone/metabolism , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Spectrometry, Fluorescence , Structure-Activity Relationship , TryptophanABSTRACT
A recombinant parathyroid hormone analog, rPTH(1-34*), was obtained from Escherichia coli using a gene polymerization strategy. The PTH gene polymer contains up to 8 copies of the gene, each separated by a cleavable linker. The polymer was expressed at very high levels and formed inclusion bodies which could be easily isolated by low-speed centrifugation. A polyhistidine leader peptide allows rapid purification via nickel chelation chromatography of the PTH polymer solubilized from the inclusion bodies. Yields of greater than 500 mg/liter have been obtained. After isolating the polymer, monomeric rPTH(1-34*) is released from the polymer by chemical cleavage with cyanogen bromide. Following cyanogen bromide cleavage and high-performance liquid chromatography purification, highly purified, biologically active rPTH(1-34*) is obtained at a yield of approximately 300 mg/liter. This is a general strategy for the high-level production of a variety of peptides and small proteins.
