Abstract
Purpose: To investigate Pichia pastoris expression system for producing clinically usable, high-quality dipeptidyl peptidase 4 recombinant protein.Methods: The yeast, Pichia pastoris, expression system was used for the production of the human recombinant dipeptidyl peptidase 4 as a secreted form. The full-length human dipeptidyl peptidase 4 corresponding to the amino acid 31-766 was subcloned into a Pichia pastoris expression vector, pPICZ □ , and transformed to Pichia pastoris X33 cells.Results: The human recombinant dipeptidyl peptidase 4 protein was expressed enzymatically as active human rDPP4(31-766) as secreted form in the yeast P. pastoris, purified and monitored its biological activity. The test DPP4 recombinant protein induced a significant increase of DDP4 activity at 10, 20 and 30 min incubation time (p < 0.05) and at 40 min (p < 0.001). A similar pattern was found for the commercial (standard) DPP4 protein at 10, 20 and 30 min (p < 0.05) and at 40 min (p < 0.001). The high standard deviation (SD) associated with the mean value for the DPP4 activity is due to incubation time sometimes associated with high DPP4 values. The values were much higher than in other groups as expected.Conclusion: Human recombinant dipeptidyl peptidase 4(31-766) protein in the yeast Pichia pastoris, obtained using the technique employed in this study can further improve production efficiency and costs of human recombinant dipeptidyl peptidase 4 and other recombinant proteins.Keywords: DPP4, Pichia pastoris, Recombinant protein, Expression, Purification
Highlights
Dipeptidyl peptidase 4 (DPP4/DPPIV/CD26 or ADAbp) is a 220 kDa homodimeric, type II transmembrane glycoprotein and a cell surface protease belonging to the prolyloligopeptidase family
DPP4 gene was amplified by RT-PCT and subcloned into the pPICZαvecter, where it is under the control of the AOX1 promoter (Fig. 1B and 1C), at its N-terminus it has α–factor signal sequence for efficient secretion into the culture medium and it contains six histidine residues for detection and purificationat its C-terminus
Specific inhibition of DPP4 dipeptidyl peptidase activity increases the half-life of the incretin hormones, glucagon-like peptide-1 and gastric inhibitory polypeptide, both involved in insulin secretion
Summary
Dipeptidyl peptidase 4 (DPP4/DPPIV/CD26 or ADAbp (adenosine deaminase binding protein)) is a 220 kDa homodimeric, type II transmembrane glycoprotein and a cell surface protease belonging to the prolyloligopeptidase family. In this study, having established the suitability of P. pastoris for the expression of the human recombinant DPP4(31-766) (human rDPP4(31-766)) as a secreted form, we set out to investigate the possibilities of producing the useful proteins having the correctly, post-translational modified properties for both basic laboratory research and industrial manufacture. The XhoI and NotI sites allow the direct insertion of the human DPP4(31-766) into pPICZα, resulting in a fusion to the α-mating factor secretion signal Resolved proteins were transferred to a nitrocellulose membrane (S&S, Dassel, Germany), blocked with 5 % nonfat dry milk in Tris-buffered saline, and probed with specific antibody against human DPP4 (DPPIV (Cell Signaling Technology, Beverly, MA, USA) followed by the secondary antibody coupled to horseradish peroxidase (Bio-Rad Laboratories, Inc).
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