Design and implementation of a high yield production system for recombinant expression of peptides

Abstract

Background: Making 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.

Results: In 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.

Conclusions: This study provides a system suitable for recombinant production of peptides for scientific research, including biological assays.

Figure 1

Schematic diagram of the expression vector pET31HT. Green: Polyhistidine tag coding sequence; red: ketosteroid isomerase gene; purple: thrombin recognition site coding sequence; light blue: cloning site restriction enzymes. Start and stop translation codons are crowned in brown.

Figure 2

SDS-PAGE analysis of recombinant protein expression in E. coli BL21(DE3). SDS-PAGE 12.5% T, 3% C; Tris-Glycine buffer system. M: Molecular weight marker; 1: KSI (14.73 kDa); 2: KSI-p53pAnt (19.09 kDa); 3: KSI-PNC27 (18.69 kDa).

Figure 3

Thrombin cleavage reaction progress at 23°C and 0.35 units of enzyme per mg of protein. A: Reaction curve. The arrow indicates addition of an equal amount of thrombin. B: SDS-PAGE 10% T, 3% C; Tris-Tricine buffer system. Different species are indicated as K-P: KSI-peptide fused peptide; K: KSI partner protein; P: Peptide. M: Molecular weight marker; lanes 1–9: samples of the reaction at different times in hours. Lane 1: 0 h; lane 2: 2 h; lane 3: 4 h; lane 4: 8 h; lane 5: 20 h; lane 6: 30 h; lane 7: 48 h; lane 8: 72 h; lane 9: 96 h.

Figure 4

Purified p53pAnt peptide. SDS-PAGE 10% T, 3% C; Tris-Tricine buffer system. M: Molecular weight marker; 1: p53pAnt. A: 0.125 μg of peptide visualized with silver staining. Blue arrow indicates a contaminant protein band. B: 2 μg of peptide visualized with Coomasie staining. The contaminant protein band is not visualized.

Figure 5

MALDI-TOF mass spectrum of the purified p53pAnt peptide.