Generation of bacterial strains for production of therapeutic human peptide hormone

Oksana Degtjarik¹, Martina Hladikova¹, Zuzana Chrastilova^2,3 and Jost Ludwig^1,4

¹University of South Bohemia, Department of Physical Biology, Zamek 136, 373 33 Nove Hrady, Czech Republic

²Zentiva a.s., U Kabelovny 130, 102 37 Praha 10, Czech Republic
³Institute of Chemical Technology, Department of Biochemistry and Microbiology, Technicka 3, 166 28 Praha 6, Czech Republic

⁴University of Bonn, IZMB / MolekulareBioenergetik, Kirschallee 1, D-53115 Bonn, Germany

Therapeutically active peptides and proteins (biopharmaceuticals or biodrugs) represent a rapidly growing proportion of marketed drugs and have an undisputed place alongside many therapies; for certain indications they even are the only effective therapy. Therapeutic peptides and proteins interact with targets that are not accessible for small chemistry-based molecules. Biopharmaceuticals cover many therapeutic areas including treatment of cancer, autoimmune diseases, diabetes, anemia, disorders associated with lack of certain proteins (e.g. human growth hormone) and others.

Parathyroid hormone (PTH) is a peptide hormone secreted by the parathyroid glands that consists of 84 amino acid residues. In human it regulates calcium and phosphate metabolism. A C-terminal truncated version consisting of the 34 first amino acids retains the biological activity.

The aim of this work was to generate a high-level expression system for production of recombinant human parathyroid hormone which eventually can be used for pharmaceutical purposes.

Because it is known, that short peptides are difficult to express in Escherichia coli, several different strategies were used to obtain suitable expression constructs for production of PTH in bacteria. One method relies on the use of fusion partners (glutathione S-transferase (GST), maltose binding protein (MBP) and others). By including an appropriate protease recognition sequence, the peptide can be separated from the fusion partner by proteolytic cleavage. Another method involves gene polymerization. Here, the gene of interest is expressed and purified as polymer and subsequently cleaved into monomers. A third approach is to express the target gene in a bacterial strain exhibiting low proteolytic activity what should also lead to higher yields of the produced peptide.

We have developed several bacterial expression systems, using all three approaches mentioned for yield enhancement. As an alternative to bacterial expression, we are also developing a yeast expression system, producing and secreting the desired peptide.

Acknowledgement: This work is supported by grants FRVS 1069/2008/G4, MSM 6046137305 and MPO 2A-2TP1/030.