Endogenous signal peptides in recombinant protein production by Pichia pastoris: From in-silico analysis to fermentation


Massahi A., ÇALIK P.

JOURNAL OF THEORETICAL BIOLOGY, cilt.408, ss.22-33, 2016 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 408
  • Basım Tarihi: 2016
  • Doi Numarası: 10.1016/j.jtbi.2016.07.039
  • Dergi Adı: JOURNAL OF THEORETICAL BIOLOGY
  • Sayfa Sayıları: ss.22-33

Özet

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 (P-GAP) 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 alpha-mating factor prepro sequence (alpha-MF) were compared. With respect to the SP23 which is 73 amino acids shorter in length compared to alpha-MF, in high cell density cultures, where carbon and energy source are limited, the substitution of SP23 for alpha-MF seems promising. alpha-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. (C) 2016 Elsevier Ltd. All rights reserved.