Synthesis of N-Glycosylated Soluble Fas Ligand.

Controlled cell death is essential for the regulation of the immune system and plays a role in pathogen defense. It is often altered in pathogenic conditions such as cancer, viral infections and autoimmune diseases. The Fas receptor and its corresponding membrane-bound ligand (FasL) are part of the extrinsic apoptosis pathway activated in these cases. A soluble form of FasL (sFasL), produced by ectodomain shedding, displays a diverse but still elusive set of non-apoptotic functions and sometimes even serves as a pro-survival factor. To gather more knowledge about the characteristics of this protein and the impact N-glycosylations may have, access to homogeneous posttranslationally modified variants of sFasL is needed. Therefore, we developed a flexible strategy to obtain such homogeneously N-glycosylated variants of sFasL by applying chemical protein synthesis. This strategy can be flexibly combined with enzymatic methods to introduce more complex, site selective glycosylations.

Semisynthesis of Homogeneous, Active Granulocyte Colony-Stimulating Factor Glycoforms.

Granulocyte colony stimulating factor (G-CSF) is a cytokine used to treat neutropenia. Different glycosylated and non-glycosylated variants of G-CSF for therapeutic application are currently generated by recombinant expression. Here, we describe our approaches to establish a first semisynthesis strategy to access the aglycone and O-glycoforms of G-CSF, thereby enabling the preparation of selectively and homogeneously post-translationally modified variants of this important cytokine. Eventually, we succeeded by combining selenocysteine ligation of a recombinantly produced N-terminal segment with a synthetic C-terminal part, transiently equipped with a side-chain-linked, photocleavable PEG moiety, at low concentration. The transient PEGylation enabled quantitative enzymatic elongation of the carbohydrate at Thr133. Overall, we were able to significantly reduce the problems related to the low solubility and the tendency to aggregate of the two protein segments, which allowed the preparation of four G-CSF variants that were successfully folded and demonstrated biological activity in cell proliferation assays.

Recent Advances in Peptide-Based Approaches for Cancer Treatment.

BACKGROUND: Peptide-based pharmaceuticals have recently experienced a renaissance due to their ability to fill the gap between the two main classes of available drugs, small molecules and biologics. Peptides combine the high potency and selectivity typical of large proteins with some of the characteristic advantages of small molecules such as synthetic accessibility, stability and the potential of oral bioavailability. METHODS: In the present manuscript we review the recent literature on selected peptide-based approaches for cancer treatment, emphasizing recent advances, advantages and challenges of each strategy. RESULTS: One of the applications in which peptide-based approaches have grown rapidly is cancer therapy, with a focus on new and established targets. We describe, with selected examples, some of the novel peptide-based methods for cancer treatment that have been developed in the last few years, ranging from naturally-occurring and modified peptides to peptidedrug conjugates, peptide nanomaterials and peptide-based vaccines. CONCLUSION: This review brings out the emerging role of peptide-based strategies in oncology research, critically analyzing the advantages and limitations of these approaches and the potential for their development as effective anti-cancer therapies.

Semisynthesis of Membrane-Attached Proteins Using Split Inteins.

The site-selective installation of lipid modifications on proteins is critically important in our understanding of how membrane association influences the biophysical properties of proteins as well as to study certain proteins in their native environment. Here, we describe the use of split inteins for the C-terminal attachment of lipid-modified peptides to virtually any protein of interest (POI) via protein trans-splicing (PTS). To achieve this, the protein of interest is expressed in fusion with the N-terminal split intein segment and the C-terminal split intein segment is prepared by solid phase peptide synthesis. A synthetic peptide carrying two lipid chains is also made chemically to serve as a membrane anchor and subsequently linked to the C-terminal split intein by native chemical ligation. Proteins of interest for our work are the prion protein as well as small GTPases; however, extensions to other POIs are possible. Detailed information for the C-terminal introduction of a lipidated membrane anchor (MA) peptide using split intein systems from Synechocystis spp. and Nostoc punctiforme for the Prion protein (PrP, as a challenging protein of interest) and the enhanced green-fluorescent protein (eGFP, as an easily trackable target protein) are provided here.