High-level production of recombinant human parathyroid hormone 1-34

Abstract

Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli beta-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein beta G-117S4HPT, constructed from amino acids 1 to 117 of beta-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.

FIG. 1

Expression vectors for the hPTH(1-34) fusion protein production. A schematic representation of the expression vectors of the partner peptides, linker peptides, and fusion proteins is given. “m” and “n” indicate the number of inserted oligohistidine linkers and the number of histidine residues, respectively. Plac, E. coli lac operator-promoter; Ttrp, E. coli trp terminator; pBR ori, replication origin of pBR322; Tet, tetracycline resistance gene; Sma I, the SmaI site diminished by linker insertion.

FIG. 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the productivity of the fusion proteins. Aliquots of the total lysate of each strain containing the same number of cells were loaded onto a sodium dodecyl sulfate–16% polyacrylamide gel (TEFCO, Chino, Japan) as described previously (11). The identity of each lane number, with the putative isoelectric point (pI_cal), is as indicated. The positions of the molecular mass standards in kilodaltons are shown to the left of the figure. pI_cal was calculated with the DNASIS program (Hitachi Tokyo, Japan).

FIG. 3

Site-specific processing of βG-117S4HPT with Kex2-660 as represented by the high-performance liquid chromatography elution profiles. Peaks: 1, hPTH(1-34); 2, β-Gal-117S-4H; 3, βG-117S4HPT. Traces: A, before processing; B, after processing.