Modifications in the Kex2 P1 ’ cleavage site in the α-MAT secretion signal lead to higher production of human granulocyte colony-stimulating factor in Pichia pastoris

AbstractThe 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 inEscherichia coli. The methylotrophic unicellular yeastPichia 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 inP. pastoris, the present study examined modification of theSaccharomyces cerevisiae derived α-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 putt ing these mutations against rarely occurring methanol slow utilizationP. 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.
Source: World Journal of Microbiology and Biotechnology - Category: Microbiology Source Type: research