SUPERVISOR: Gerald STRIEDNER

PROJECT ASSIGNED TO: Martin GIBISCH

High molecular weight biomolecules have so far dominated the field of protein-derived therapeutics in many areas. Since recent years, however, peptides (proteins <100 amino acids) have gained great attention for many therapeutic applications and are considered next-level biopharmaceuticals with already over 50 peptide-associated drugs on the market [1]. Given their small size and (relatively) low molecular weight, peptides have certain advantages over large proteins and antibodies. Among others, these include a high potency of action, wide range of targets, low toxicity, fewer side effects, and a low accumulation in tissues [2]. The use of peptides covers a broad range of applications, most notably cancer therapy (pore forming peptide can induce necrosis or apoptosis [3]), antimicrobials (disrupting the function of bacterial cell membranes [4]), as well as targeted cargo delivery (DNA, siRNA, plasmids,…) by CPPs (cell-penetrating peptides), Alzheimer’s disease, Malaria, and signaling (peptide hormones such as insulin and parathyroid hormone) [2], [4], [5]. Peptides are commonly extracted from biological material or chemically synthesized [5], [6]. Both current state-of-the-art methods for peptide production, however, rely on the availability of biomaterial for extraction and heavy use of hazardous agents/solvents during complex chemical processes, which often are limited (yield-dependent) to the synthesis of peptides up to 70 amino acids [6], [7]. Recombinant expression in Escherichia coli could potentially circumvent these issues and offers a platform for efficient production of low molecular weight peptides.

In this thesis, four different model peptides will be expressed in E. coli. The peptides will be translocated to the periplasm to favor proper formation of disulfide bonds, thus correct folding. Peptide expression, folding, and activity will be analyzed using various analytical methods. Furthermore, outer membrane permeability of the host organism(s) will be adjusted in-process using moleculobiological tools. Peptides should leak out of the periplasm into the medium to simplify downstream processing. Membrane permeability will be characterized via atomic force microscopy and other analytical assays. The goal is to quantify and link outer membrane permeability to different mechanical properties of the cell. Overall, biologically active peptides should be produced extracellularly in soluble form by adjusting the membrane permeability of growing and producing cell.

This project is part of the CD Laboratory for production of next-level biopharmaceuticals in E. coli and funded by the Christian Doppler Forschungsgesellschaft and Boehringer Ingelheim RCV GmbH & Co KG.

Literature

[1]    A. Henninot, J. C. Collins, and J. M. Nuss, “The Current State of Peptide Drug Discovery: Back to the Future?,” Journal of Medicinal Chemistry, vol. 61, no. 4. American Chemical Society, pp. 1382–1414, Feb. 22, 2018, doi: 10.1021/acs.jmedchem.7b00318.

[2]    S. Marqus, E. Pirogova, and T. J. Piva, “Evaluation of the use of therapeutic peptides for cancer treatment,” J. Biomed. Sci., vol. 24, no. 1, pp. 1–15, Mar. 2017, doi: 10.1186/s12929-017-0328-x.

[3]    R. J. Boohaker, M. W. Lee, P. Vishnubhotla, J. L. M. Perez, and A. R. Khaled, “The Use of Therapeutic Peptides to Target and to Kill Cancer Cells,” Curr. Med. Chem., vol. 19, no. 22, pp. 3794–3804, Jul. 2012, doi: 10.2174/092986712801661004.

[4]    S. Lien and H. B. Lowman, “Therapeutic peptides,” Trends in Biotechnology, vol. 21, no. 12. Elsevier Ltd, pp. 556–562, Dec. 01, 2003, doi: 10.1016/j.tibtech.2003.10.005.

[5]    J. L. Lau and M. K. Dunn, “Therapeutic peptides: Historical perspectives, current development trends, and future directions,” Bioorganic Med. Chem., vol. 26, no. 10, pp. 2700–2707, Jun. 2018, doi: 10.1016/j.bmc.2017.06.052.

[6]    A. Isidro-Llobet et al., “Sustainability Challenges in Peptide Synthesis and Purification: From R&D to Production,” J. Org. Chem., vol. 84, no. 8, pp. 4615–4628, Apr. 2019, doi: 10.1021/acs.joc.8b03001.

[7]    W. C. Chan and P. D. White, Fmoc solid phase peptide synthesis: A practical apporach. Oxford University Press, 2000.