title: Endogenous signal peptides in recombinant protein production by Pichia pastoris: From in-silico analysis to fermentation.
abstract: For extracellular recombinant protein production, the efficiency of five endogenous secretion signal peptides (SPs) of Pichia pastoris, SP13 (MLSTILNIFILLLFIQASLQ), SP23 (MKILSALLLLFTLAFA), SP24 (MKVSTTKFLAVFLLVRLVCA), SP26 (MWSLFISGLLIFYPLVLG), SP34 (MRPVLSLLLLLASSVLA), selected based on their D-score which quantifies the signal peptide-ness of a given sequence segment, was investigated using recombinant human growth hormone (rhGH) as the model protein. The expression was conducted under glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). The highest secretion efficiency among endogenous SPs was obtained by SP23 followed by SP24, SP34, SP13 and SP26, respectively. The fermentation characteristics of rhGH production by the use of SP23, the most favorable endogenous SP of P.pastoris, and Saccharomyces cerevisiae α-mating factor prepro sequence (α-MF) were compared. With respect to the SP23 which is 73 amino acids shorter in length compared to α-MF, in high cell density cultures, where carbon and energy source are limited, the substitution of SP23 for α-MF seems promising. α-MF higher secretion efficiency was verified by major physicochemical properties including hydropathy index, isoelectric point, and aliphatic index. Regarding the examined endogenous SPs, there was no clear correlation between secretion efficiency and major physicochemical properties when each of these properties was considered independently. To find a correlation, factors such as protein N-terminus effect, length of the SP, secondary structure of the SP, and interactions of the selected properties should also be investigated.
authors.affiliation: Industrial Biotechnology and Metabolic Engineering Laboratory, Chemical Engineering Department, Middle East Technical University, 06800 Ankara, Turkey; Department of Biotechnology, Graduate School of Natural and Applied Sciences, Middle East Technical University, 06800 Ankara, Turkey.
authors.forename: Aslan
authors.initials: A
authors.lastname: Massahi
authors.affiliation: Industrial Biotechnology and Metabolic Engineering Laboratory, Chemical Engineering Department, Middle East Technical University, 06800 Ankara, Turkey; Department of Biotechnology, Graduate School of Natural and Applied Sciences, Middle East Technical University, 06800 Ankara, Turkey. Electronic address: pcalik@metu.edu.tr.
authors.forename: Pınar
authors.initials: P
authors.lastname: Çalık
mesh_terms: D000595:Amino Acid Sequence; D003198:Computer Simulation; D005285:Fermentation; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+) / Q000235:genetics; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072235:Mating Factor; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D000072235:Mating Factor; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+)
keywords: Endogenous signal peptide; GAP promoter; Human growth hormone; Pichia pastoris; Secretion