| Title: | Differential role of segments of alpha-mating factor secretion signal in Pichia pastoris towards granulocyte colony-stimulating factor emerging from a wild type or codon optimized copy of the gene |
| Address: | "Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi, 110016, India. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi, 110016, India. saroj98@hotmail.com" |
| DOI: | 10.1186/s12934-020-01460-8 |
| ISSN/ISBN: | 1475-2859 (Electronic) 1475-2859 (Linking) |
| Abstract: | "BACKGROUND: The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. RESULTS: Mutations in the pro-region of the alpha-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Delta57-70 type, led to highest G-CSF titre of 39.4 +/- 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30-43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matalpha:Delta47-49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Delta30-43 in the WT-GCSF was partially restored (by 60%) when the Delta57-70 was added. The effect of Delta47-49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Delta57-70 indicating the importance of the 47-49 region. A stimulatory effect of Delta57-70 was confirmed in the double deletion (Matalpha:Delta57-70;47-49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Delta57-70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. CONCLUSIONS: Among the different truncations (Matalpha:Delta57-70, Matalpha:Delta47-49, Matalpha:Delta30-43, Matalpha:Delta57-70;30-43, Matalpha:Delta57-70;47-49) in pro-peptide of alpha-MAT secretion signal, Matalpha:Delta57-70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matalpha truncations. On the other hand, Matalpha:Delta30-43 and Matalpha:Delta47-49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein" |
| Keywords: | "5' Untranslated Regions/genetics Codon/*genetics Computational Biology Granulocyte Colony-Stimulating Factor/*biosynthesis Humans Mating Factor/*genetics Mutation Pichia/genetics/*metabolism Protein Structure, Secondary Recombinant Proteins/biosynthesis S;" |
| Notes: | "MedlineAggarwal, Sakshi Mishra, Saroj eng England 2020/10/31 Microb Cell Fact. 2020 Oct 29; 19(1):199. doi: 10.1186/s12934-020-01460-8" |
