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Prolong the peptide bioactivity by reversible self-assembly | LifeTein Peptide Blog

Most of the potential therapeutic peptides have low solubility, chemical instability or low stability against protease. So it is essential to modify and optimize the peptides to improve the peptide bioavailability. One novel approach is to create a self-assemb

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Most of the potential therapeutic peptides have low solubility, chemical instability or low stability against protease. So it is essential to modify and optimize the peptides to improve the peptide bioavailability.

One novel approach is to create a self-assembly, highly ordered, and stable nanostructure. For instance, many peptide hormones including glucagon are stored in the form of β-sheet rich amyloid-like fibrils via a hydrogen bond network in the secretory cell.

The oxyntomodulin is a peptide with a potential to treat obesity and diabetes. It is a 37-amino-acid proglucagon-derived peptide hormone with sequence homology to both glucagon and glucagon-like peptide-1 (GLP-1). The oxyntomodulin peptide self-assembles into a stable nanofibril formulation and later on releases an active peptide.

Here is the sequence of human oxyntomodulin: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala, HSQGTFTSDY SKYLDSRRAQ DFVQWLMNTK RNRNNIA.

Oxyntomodulin self-assembles into fibrillar nanostructures

The peptide concentration was 10 mg/mL in water at an incubation pH of between 7.0 and 7.3 and low ionic strength (0.09% saline).

The solution was incubated at 37 °C and agitated by orbital shaking.

After incubation for five days, the solution turned turbid due to the formation of a suspension of aggregates. The conversion yield of the self-assembly of Oxyntomodulin into fibrillar nanostructures was estimated to be 99% under these conditions.

The nanofibrils were next used to seed a solution of free peptide at 10 mg/mL in water. 5. The solution was incubated without agitation for one week and then diluted to 1 mg/mL in 0.09% saline for another 2–9 days of incubation at 37 °C and then nine days at room temperature.

Free peptides were mainly in an α-helical conformation. The fibrillar Oxm showed the majority of β-sheet and some α-helical content.

Peptide nanofibrils dissociate to release intact peptide

1 mg/mL nanofibrils were incubated in water. 37% of the peptide was released after four hours of incubation.

In aqueous HCl, a 77% release was observed after only four hours. The peptide remained chemically intact after discharge from the nanofibrils

Benefits:

The released peptide is active and nontoxic in vitro.

The nanofibrils prolong peptide serum bioactivity in vivo. And there is no need to engineer or modify the original peptide. Other peptide hormones including glucagon, GLP-1, exendin-4, calcitonin, and gastric inhibitory peptide, are known to self-assemble. The self-assembly method may be used for the clinical application of reversibly self-assembling nanofibrils.

Reference:Controlling the bioactivity of a peptide hormone in vivo by reversible self-assembly. Nature Communications, volume 8, Article number: 1026 (2017)

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Studying Cell-Cell Interactions with LIPSTIC

In immunology research, Cy7-labelled LPETGG peptides have proven instrumental in the LIPSTIC (Labelling Immune Partnerships by SorTagging Intercellular Contacts) technique. This elegant method uses bacterial sortase A to enzymatically transfer fluorescent dyes from the LPETGG substrate onto interacting cell surfaces, enabling researchers to track dynamic immune partnerships in vivo and in vitro with single-cell resolution. Such applications underscore the versatility of Cy7 beyond simple structural labelling.

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Clinical Trials and Research

Several clinical trials have explored the use of PADRE in cancer vaccines. For instance, vaccines targeting Mucin 1 (MUC1), a glycoprotein overexpressed in many cancers, have shown promising results when combined with PADRE. These vaccines have demonstrated the ability to elicit strong immune responses, including the production of antibodies against cancer-specific antigens.

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Practical and safety references

These excerpts are educational, not personalised medical instructions.

How-to reference

How to solubilize my synthetic peptides? #

Please refer to this FAQ for details: Handling and Storage of Synthetic Peptides. If the peptides are still cloudy, or turbid, you may have reached the limit of solubility. When the peptides are insoluble in the buffer, please try to sonicate, centrifuge, and lyophilize the peptide. Make sure to break the lyophilized lumps into a fine powder. Then try a small volume of a good agent 8M Urea, NMP, DMF, or DMSO to dissolve the peptide. Then dilute with water or your desired buffer. For peptides with Arg or LYs, you should try to lower the pH to 6 because the protonated amino acids will help solubility. Sonication and the following solvents may help with difficult peptides: 1) Begin with 100 % acetonitrile then dilute with water until 50% 2) Begin with 100% DMSO then dilute with water until 30 % 3) Dissolve it with 8M Urea 4) Dissolve it with 6 or 8 M Guanidine hydrochloride 5) 6M GuHCL, 0.05% TFA, pH2, 6) 100% TFA 7) 40% AcOH, 30%ACN, 30% water

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Storage reference

Storage temperatures and conditions #

For many of our antibodies, freezing at -20 C or -80 C in small aliquots is the optimal storage condition. Aliquotting minimizes damage due to freezing and thawing, as well as contamination introduced by pipetting from a single vial multiple times. Aliquots should be no smaller than 10 µl. Upon receiving the antibody, centrifuge at 5,000 x g for 30 seconds to pull down the solution, and transfer aliquots into low-protein-binding microcentrifuge tubes. Antibodies should be frozen as soon as possible, storage at 4 C upon receipt of the antibody is acceptable for one to two weeks, followed by freezing for long-term storage. To prevent microbial contamination, sodium azide can be added to an antibody preparation to a final concentration of 0.02% (w/v). If using antibodies for in vivo studies, please be sure to use preparations that do not contain sodium azide. This antimicrobial agent blocks the cytochrome electron transport system. Sodium azide will interfere with any conjugation that involves an amine group and should be removed before proceeding with the conjugation. After conjugation, antibodies can be stored in sodium azide but 0.01% thimerosal (Merthiolate), which does not have a primary amine, is an acceptable alternative. Sodium azide can be removed from antibody solutions by dialysis or gel filtration. The molecular weight of IgG is 150,000 daltons (IgM is ~ 600,000); the molecular weight of sodium azide is 65 daltons. A micro-dialysis unit with a cut off at 14,000 dalt…

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