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

Massahi, Aslan
Çalık, Pınar
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.


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ÖZDAMAR, HASAN TUNÇER; Sentuerk, Birguel; Yilmaz, Oezge Deniz; Calik, Guezide; Celik, Eda; Çalık, Pınar (2009-01-01)
We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre-) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg-promoter in B. subtilis. Recombinant-hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-termina...
Design and construction of double promoter systems and their use in pharmaceutical protein production in P. Pastoris
Demir, İrem; Çalık, Pınar; Department of Chemical Engineering (2019)
Intracellular phenomena such as promoter strength, mRNA secondary structure, translation efficiency and codon preference, 5′-untranslated region processing, and protein turnover, have impacts directly on the expression of heterologous genes. Design of multi-promoter expression systems with constituent strong promoters and engineered promoter variants is a novel metabolic engineering strategy for increasing the promoter strength further, and tuning the expression for recombinant protein (r-protein) productio...
Metabolic reaction network of Pichia pastoris with glycosylation reactions: Flux analysis for erythropoietin production
Eskitoros, Melda S.; Ata, Ozge; Çalık, Pınar (Wiley, 2014-11-01)
BACKGROUND: Biochemical reaction network of Pichia pastoris was improved by including N-glycosylation pathway reactions to determine the intracellular reaction rates for glycosylated protein production.
Metabolic engineering with a novel promoter in Pichia pastoris for recombinant human growth hormone production: effects of oxygen transfer conditions
Kalender, Özge; Çalık, Pınar; Özdamar, Tunçer; Department of Chemical Engineering (2018)
The objectives of this thesis are investigation of, i) the effects of oxygen transfer conditions on recombinant protein production in Pichia pastoris strains designed with novel naturally occuring pyruvate kinase (PYK) promoter (PPYK) which is a potential promoter for recombinant protein production under low to moderate oxygen transfer conditions, and ii) influences of engineering with single- and multi- copy genes, in fed-batch fermentation processes. Production of recombinant human growth hormone (rhGH) b...
In-silico determination of Pichia pastoris signal peptides for extracellular recombinant protein production
Massahi, Aslan; Çalık, Pınar (2015-01-07)
In-silico identified novel secretory signal peptides (SPs) are required in vivo to achieve efficient transfer or to prevent other cellular proteins from interfering with the process in extracellular recombinant protein (r-protein) production. 56 endogenous and exogenous secretory SPs, have been used or having the potential to be used in Pichia pastoris for r-protein secretion, were analyzed in-silico using the softwares namely SignalP4.1, Phobius, WolfPsort0.2, ProP1.0, and NetNGlyc1.0. Among the predicted ...
Citation Formats
A. Massahi and P. Çalık, “Endogenous signal peptides in recombinant protein production by Pichia pastoris: From in-silico analysis to fermentation,” JOURNAL OF THEORETICAL BIOLOGY, pp. 22–33, 2016, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/36194.