Impact of N-Terminal PEGylation on Synthesis and Purification of Peptide-Based Cancer Epitopes for Pancreatic Ductal Adenocarcinoma (PDAC).

Peptide-based cancer vaccines have shown promising results in preclinical trials focusing on tumor immunotherapy. However, the presence of hydrophobic amino acid segments within these peptide sequences poses challenges in their synthesis, purification, and solubility, thereby hindering their potential use as cancer vaccines. In this study, we successfully synthesized peptide sequences derived from mesothelin (MSLN), a tumor-associated antigen overexpressed in pancreatic ductal adenocarcinoma (PDAC) by conjugating them with monodisperse polyethylene glycol (PEG). By PEGylating mesothelin epitopes of varying lengths (ranging from 9 to 38 amino acids) and hydrophobicity (60-90%), we achieved an effective method to improve the peptide yield and facilitate the processes of synthesis and purification. PEGylation significantly enhanced the solubility, facilitating the single-step purification of lengthy hydrophobic peptides. Most importantly, PEGylation did not compromise cell viability and had little to no effect on the immunogenicity of the peptides. In contrast, the addition of a palmitoyl group to increase immunogenicity led to reduced yield and solubility. Overall, PEGylation proves to be an effective technique for enhancing the solubility and broadening the range of utility of diverse long hydrophobic peptides.

Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine?

The deprotection step is crucial in order to secure a good quality product in Fmoc solid phase peptide synthesis. 9-Fluorenylmethoxycarbonyl (Fmoc) removal is achieved by a two-step mechanism reaction favored by the use of cyclic secondary amines; however, the efficiency of the reaction could be affected by side reactions and by-product formation. Several aspects have to be taken into consideration when selecting a deprotection reagent: its physicochemical behavior, basicity (pKa) and polarity, concentration, and time of reaction, toxicity and disposability of residues and, finally, availability of reagents. This report presents a comparison of the performance of three strategies for deprotection using microwave-assisted Fmoc peptide synthesis. Four peptide sequences were synthesized using Rink amide resin with a Liberty Blue™ automated synthesizer and 4-methylpiperidine (4MP), piperidine (PP), and piperazine (PZ) as Fmoc removal reagents. In the first instance all three reagents behaved similarly. A detailed analysis showed a correlation between the hydrophobicity and size of the peptide with the yield and purity of the obtained product. The three reagents are interchangeable, and replacement of piperidine could be advantageous regarding toxicity and reagent handling.

Hepcidin, Cathelicidin-1 and IL-8 as immunological markers of responsiveness in early developmental stages of rainbow trout.

During the early developmental stage of salmonids, high mortality occurs largely as a result of pathogens. These cause low immune competence in fry, producing disease, decreasing production and finally leading to economic losses. Therefore, the aim of this study was to characterise the developmental stages in which rainbow trout acquires immune response capability when challenged with LPS from Pseudomona aeruginosa for 8 h, studying the hepcidin, cathelicidin-1 and IL-8. Total RNA was extracted from fry at 34, 42, 56 and 66 days post hatching (dph). Hepcidin and cathelicidin-1 transcripts were detected only at days 34 and 42, whereas the IL-8 transcript was detected from day 34 to day 66. To analyse the protein expression in the fry, polyclonal anti-peptide antibodies were generated in rabbit. These three immune sera demonstrated the ability to recognise the whole molecule in biological samples. Immunofluorescence showed that skin, gills and intestine mainly responded to the LPS challenge, indicating that these portals of pathogen entry are capturing LPS. This study constitutes a valuable approach, since it has the potential to identify molecules with biological activity that can be used to evaluate the status of fry in culture.