Abstract
BackgroundCalreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca2+ 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.ResultsExpression 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.ConclusionsYeasts 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0356-8) contains supplementary material, which is available to authorized users.
Highlights
Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca2+ homeostasis
Expression and purification of human CRT from yeasts S. cerevisiae and P. pastoris from culture medium Our results show that expression of a full-length human CRT precursor including its native signal sequence resulted in a high-level secretion of the recombinant protein into the culture medium by two yeast species, S. cerevisiae and P. pastoris
In the S. cerevisiae system, we expressed the gene encoding CRT under control of the yeast PGK1 gene promoter in the same vector, pFDC [20], that was previously used for the expression of secreted human BiP and ERp57 in yeast [21, 22] and show that CRT was secreted into the culture medium (Fig. 1)
Summary
Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca2+ 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. CRT functions in the ER as a calcium-binding chaperone involved in a variety of biological processes including quality control of protein folding [1,2,3], regulation of calcium homeostasis [2, 4, 5] and MHC class I antigen processing [6, 7]. Future fundamental, applied and therapeutic studies and use will likely require large amounts of affordable high-quality recombinant human CRT protein with native functional capacity, insofar as possible
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