| Title: | Modifications in the Kex2 P1' cleavage site in the alpha-MAT secretion signal lead to higher production of human granulocyte colony-stimulating factor in Pichia pastoris |
| Address: | "Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi, 110016, India. International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New-Delhi, 110067, India. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi, 110016, India. saroj98@hotmail.com" |
| Journal Title: | World J Microbiol Biotechnol |
| DOI: | 10.1007/s11274-021-03167-3 |
| ISSN/ISBN: | 1573-0972 (Electronic) 0959-3993 (Linking) |
| Abstract: | "The human granulocyte colony-stimulating factor (G-CSF) is one of the hematopoietic growth factors administered for chemotherapy induced neutropenia and is currently produced through recombinant route in Escherichia coli. The methylotrophic unicellular yeast Pichia pastoris (syn. Komagataella phaffii) makes a good host for production of human therapeutics as the proteins are low-mannose glycosylated, disulfide bonded and correctly folded on their way to the cell exterior. Given the low level of production of G-CSF in P. pastoris, the present study examined modification of the Saccharomyces cerevisiae derived alpha-mating type secretory signal sequence to enhance its production. The substitution of Glu, at the P1' position of the Kex2 cleavage site, by Val/Ala led to extracellular production of ~ 60 mg/L of G-CSF in the extracellular medium. Production was further increased to ~ 100 mg/L by putting these mutations against rarely occurring methanol slow utilization P. pastoris X-33 host. Analysis of the modelled structure of the signal peptide indicated exposed loop structures, created by presence of Val/Ala, that favour cleavage by the Kex2 peptidase thereby leading to enhanced production of G-CSF. The conformational changes, induced on account of binding between the signal sequence and the cargo protein (G-CSF), also appear to play an important role in the final yield of the extracellular protein" |
| Keywords: | "Granulocyte Colony-Stimulating Factor/*biosynthesis/genetics Humans Mating Factor/*chemistry/genetics/metabolism Proprotein Convertases/genetics/*metabolism Protein Sorting Signals/*genetics Protein Structure, Secondary Recombinant Proteins/biosynthesis S;" |
| Notes: | "MedlineAggarwal, Sakshi Mishra, Saroj eng Germany 2021/10/17 World J Microbiol Biotechnol. 2021 Oct 16; 37(11):197. doi: 10.1007/s11274-021-03167-3" |
