Isotope-labelled peptides

Lead Chief Investigators: 

Gottfried Otting, The Australian National University 

Richard Payne, The University of Sydney

Summary

Site-specific labels allow site-specific interrogation of structure and function. Chemical synthesis of isotope-labelled peptides is prohibitively expensive. In vivo production of peptides is possible in fusion with larger proteins, but experience shows that the peptides tend to be lost following proteolytic cleavage, e.g. with TEV protease. Preliminary experiments by the Otting group have shown that fusions with calmodulin successfully produce a 20-residue peptide that could not be produced in fusions with trigger factor, ubiquitin, SUMO domain or maltose binding protein. This project will explore the general validity of the approach. In a next step, the isotope-labelled peptide is to be used for fusion with unlabelled polypeptide chains to produce a segmentally isotope-labelled protein. 

Relevance to the Centre

This project aligns with the Centre’s aim to decode the function of peptides.

Report: 

Aligned with the Centre’s vision to decode the biological functions of peptides, Centre Investigator Gottfried Otting and PhD researchers Yi Jiun Tan, Iresha Herath, and Dilhara Herath at the Australian National University developed a new method for producing site-specific labelled peptides using an affordable, high-yielding bacterial expression system, which will overcome the need for expensive chemical precursors and reduce the time and labour needed in synthetic chemistry. This approach aims to produce isotope-labelled peptides, using an in vivo method, as functional beacons to help biochemists navigate the molecular surface of proteins and decode their biological functions by Nuclear Magnetic Resonance (NMR) spectroscopy. 

In this work, peptides were biosynthetically produced through in vivo expression in E. coli cells. The in vivo expression of unstructured peptides is known to be susceptible to fast and premature degradation by the bacterial amino acid recycling system. The recycling system helps the bacteria to recover and reuse resources, but it does limit the amount of product we can obtain. To overcome this challenge, synthetic DNA templates were rationally designed with specific safety features to maximize product output and limit peptide recycling. For example, the templates ensured that the N-terminal end of the peptide was attached to a soluble and folded protein domain to improve the target peptide’s in vivo stability. Additionally, the protein domain was chosen to bind the target peptide to restrict accessibility to digestive enzymes. Finally, the template was programmed to release the peptide at the TEV cleavage site while avoiding any cloning artifacts. Cloning artifacts are by-products that produce misleading results during the cloning of DNA fragments. Numerous DNA templates (referred to as fusion constructs) were developed to produce peptides fused with larger proteins. Examples of larger proteins used were trigger factors, ubiquitin, SUMO domain, maltose binding protein, and calmodulin. 

Through the bacterial expression system, the team successfully produced a 43-residue peptide derived from the putative docking domain of the epoxyketone biosynthetic assembly line, and various versions of the calmodulin-binding peptide MARCKS (myristoylated alanine-rich C kinase substrate). An array of uniformly 15N-labelled peptides was produced in good quantity and high purity, sufficient for NMR analysis. Chemical synthesis of isotope-labelled peptides typically involves expensive precursors and is time consuming. In contrast, the bacterial system provides a resourceful and efficient approach to creating isotope-labelled amino acids using 15N-labelled ammonium chloride and 13C-labelled glucose precursors.  

The fusion constructs with calmodulin yielded the best results, producing the highest amount of product, demonstrating remarkable resilience to digestion, regardless of the length or charge of the peptide. This finding is important for future studies that aim to incorporate isotopically labelled molecules to specifically tag larger proteins to help biochemists navigate the molecular surface of macromolecules using NMR spectroscopy.  

To further exemplify the robustness of the bacterial expression system, peptides containing single copies of genetically encoded non-canonical amino acids were synthesized. These unnatural amino acids were provided by research fellow Dr Elwy Abdelkader, and the work was developed in collaboration with Associate Investigator Thomas Huber. The system also explored the biosynthesis of isotope-labelled peptides fused with an intein system, aiming to achieve segmental isotope labelling. The intein system is a unique tool used in synthetic biology to produce a precursor protein that is separated into three segments. The internal protein segment (called intein) undergoes self-cleavage and triggers the fusing of the two external protein segments (called extein). Isotope-labelling of the different segments can help researchers distinguish different parts of the protein under investigation.  Work is currently being pursued to increase the quantity of proteins obtained.  

CI Gottfried Otting and his team at ANU have established a new strategy to synthesize peptides with isotope labels or unnatural amino acids, which are becoming increasingly important to medicinal chemists who are working to produce designer peptides through the biosynthetic pathway, and biochemists wanting to label larger proteins with NMR-active tags. This new method uses an affordable, high-yielding bacterial expression system, which brings about a more sustainable approach to produce peptides and minimize the production of excess chemical waste. This approach also addresses the challenges of purchasing expensive precursors associated with chemical synthesis of isotope-labelled peptides. With this method on hand, CI Gottfried Otting and his group can now effectively produce unnatural peptides, which are instrumental to uncovering conformational change, peptide-protein interactions and protein dynamics using NMR spectroscopy. The applications for this project and their results have not yet been disclosed publicly. 

CIPPS contributors: Professor Gottfried Otting (ANU), Professor Greg Challis (Monash), Professor Thomas Huber (ANU), Dr Elwy Abdelkader (ANU)