High-level secretion of native recombinant human calreticulin in yeast

Evaldas Čiplys, Eimantas Žitkus, Leslie I. Gold, Julien Daubriac, Savvas C Pavlides, Peter Højrup, Gunnar Houen, Wen-An Wang, Marek Michalak, Rimantas Slibinskas

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Abstract

BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris.

RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium.

CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.

Original languageEnglish
Article number165
JournalMicrobial Cell Factories
Volume14
Issue numberOctober
Number of pages14
ISSN1475-2859
DOIs
Publication statusPublished - 2015

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Calreticulin
Yeast
Proteins
Recombinant proteins
Saccharomyces cerevisiae
Assays
Recombinant Proteins
Culture Media
human calreticulin
Electrospray ionization
Cathode ray tubes
Physiological Phenomena
Fibroblasts
Bioreactors
Batch Cell Culture Techniques
Pichia
Electrospray Ionization Mass Spectrometry
Drug products
Purification
Mass spectrometry

Cite this

Čiplys, E., Žitkus, E., Gold, L. I., Daubriac, J., Pavlides, S. C., Højrup, P., ... Slibinskas, R. (2015). High-level secretion of native recombinant human calreticulin in yeast. Microbial Cell Factories, 14(October), [165]. https://doi.org/10.1186/s12934-015-0356-8
Čiplys, Evaldas ; Žitkus, Eimantas ; Gold, Leslie I. ; Daubriac, Julien ; Pavlides, Savvas C ; Højrup, Peter ; Houen, Gunnar ; Wang, Wen-An ; Michalak, Marek ; Slibinskas, Rimantas. / High-level secretion of native recombinant human calreticulin in yeast. In: Microbial Cell Factories. 2015 ; Vol. 14, No. October.
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abstract = "BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris.RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 {\%} of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium.CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.",
author = "Evaldas Čiplys and Eimantas Žitkus and Gold, {Leslie I.} and Julien Daubriac and Pavlides, {Savvas C} and Peter H{\o}jrup and Gunnar Houen and Wen-An Wang and Marek Michalak and Rimantas Slibinskas",
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Čiplys, E, Žitkus, E, Gold, LI, Daubriac, J, Pavlides, SC, Højrup, P, Houen, G, Wang, W-A, Michalak, M & Slibinskas, R 2015, 'High-level secretion of native recombinant human calreticulin in yeast', Microbial Cell Factories, vol. 14, no. October, 165. https://doi.org/10.1186/s12934-015-0356-8

High-level secretion of native recombinant human calreticulin in yeast. / Čiplys, Evaldas; Žitkus, Eimantas; Gold, Leslie I.; Daubriac, Julien; Pavlides, Savvas C; Højrup, Peter; Houen, Gunnar; Wang, Wen-An; Michalak, Marek; Slibinskas, Rimantas.

In: Microbial Cell Factories, Vol. 14, No. October, 165, 2015.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - High-level secretion of native recombinant human calreticulin in yeast

AU - Čiplys, Evaldas

AU - Žitkus, Eimantas

AU - Gold, Leslie I.

AU - Daubriac, Julien

AU - Pavlides, Savvas C

AU - Højrup, Peter

AU - Houen, Gunnar

AU - Wang, Wen-An

AU - Michalak, Marek

AU - Slibinskas, Rimantas

PY - 2015

Y1 - 2015

N2 - BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris.RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium.CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.

AB - BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris.RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium.CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.

U2 - 10.1186/s12934-015-0356-8

DO - 10.1186/s12934-015-0356-8

M3 - Journal article

C2 - 26471510

VL - 14

JO - Microbial Cell Factories

JF - Microbial Cell Factories

SN - 1475-2859

IS - October

M1 - 165

ER -