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Microb Cell Fact. 2015 Oct 15;14:165. doi: 10.1186/s12934-015-0356-8.

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

Author information

1
Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.A. Graičiūno 8, 02241, Vilnius, Lithuania. evaldas.ciplys@bti.vu.lt.
2
Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.A. Graičiūno 8, 02241, Vilnius, Lithuania. eimantas.zitkus@bti.vu.lt.
3
Division of Translational Medicine, Department of Medicine, New York University School of Medicine, 550 First Avenue, NB17E4, New York, NY, 10016, USA. leslie.gold@nyumc.org.
4
Division of Translational Medicine, Department of Medicine, New York University School of Medicine, 550 First Avenue, NB17E4, New York, NY, 10016, USA. julien.daubriac@nyumc.org.
5
Division of Translational Medicine, Department of Medicine, New York University School of Medicine, 550 First Avenue, NB17E4, New York, NY, 10016, USA. savvaspavlides@gmail.com.
6
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark. php@bmb.sdu.dk.
7
Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, 2300, Copenhagen, Denmark. gh@ssi.dk.
8
Department of Biochemistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada. wenan@ualberta.ca.
9
Department of Biochemistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada. marek.michalak@ualberta.ca.
10
Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, V.A. Graičiūno 8, 02241, Vilnius, Lithuania. rimantas.slibinskas@bti.vu.lt.

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.

PMID:
26471510
PMCID:
PMC4608220
DOI:
10.1186/s12934-015-0356-8
[Indexed for MEDLINE]
Free PMC Article

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