Abstract
BackgroundMaking peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome. A non common but interesting research area is related to peptides with intracellular targets, which opens up new possibilities, allowing the modulation of processes occurring within the cell or interference with signaling pathways. However, if the bioactive sequence requires fusion to a carrier peptide to allow access into the cell, the resulting peptide could be such a length that traditional production could be difficult. The goal of the present study was the development of a flexible recombinant expression and purification system for peptides, as a contribution to the discovery and development of these potentially new drugs.ResultsIn this work, a high throughput recombinant expression and purification system for production of cell penetrating peptides in Escherichia coli has been designed and implemented. The system designed produces target peptides in an insoluble form by fusion to a hexahistidine tagged ketosteroid isomerase which is then separated by a highly efficient thrombin cleavage reaction procedure. The expression system was tested on the anticancer peptides p53pAnt and PNC27. These peptides comprise the C-terminal region and the N-terminal region of the protein p53, respectively, fused by its carboxyl terminal extreme to the cell penetrating peptide Penetratin. High yields of purified recombinant fused peptides were obtained in both cases; nevertheless, thrombin cleavage reaction was successful only for p53pAnt peptide release. The features of the system, together with the procedure developed, allow achievement of high production yields of over 30 mg of highly pure p53pAnt peptide per g of dry cell mass. It is proposed that the system could be used for production of other peptides at a similar yield.ConclusionsThis study provides a system suitable for recombinant production of peptides for scientific research, including biological assays.
Highlights
Making peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome
As an alternative synthesis method, recombinant production of peptides has become a good choice for production of long peptides, mainly because it offers a good combination of cost-effectiveness, scalability and sustainability [4,5]
This paper shows the design of a system for producing high yield fused recombinant peptides and a procedure for efficient enzymatic cleavage to liberate the target peptides, which allowed production of highly pure p53pAnt peptide
Summary
Making peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome. These are cationic peptides with the ability to get into the cells and it has been demonstrated that they can mediate intracellular delivery of covalently bonded molecules, amongst them bioactive peptide sequences [8,9,10,11,12,13] In this case, the fusion usually results in a final peptide longer than 30 amino acids, making traditional production difficult. Of the peptides in Phase II studies, there are three bioactive peptides that incorporate a CPP sequence to enter the cells [7] Producing these peptides efficiently in order to minimize the production cost by means of a recombinant peptide production system, could make these peptides more attractive for the pharmaceutical industry
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
