- Open Access
High-level secretion of native recombinant human calreticulin in yeast
© Čiplys et al. 2015
- Received: 14 July 2015
- Accepted: 7 October 2015
- Published: 15 October 2015
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. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris.
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.
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.
- Human Placenta
- Yeast Culture Medium
- Sartorius Stedim Biotech
- Native Signal Sequence
- Endoplasmic Reticulum Retrieval
CRT functions in the ER as a calcium-binding chaperone involved in a variety of biological processes including quality control of protein folding [1–3], regulation of calcium homeostasis [2, 4, 5] and MHC class I antigen processing [6, 7]. In addition to these essential intracellular functions, CRT has other important functional roles outside of the ER that are critical to various physiological and disease processes, particularly stress-related disease . For example, CRT has a significant role in both the innate and adaptive immune response and cell surface CRT is required for phagocytosis of apoptotic cells . In this vein, both intracellular and extracellular CRT are important in the host immune response to cancer with respect to activation of T cells, peptide loading with tumor antigens, and in the phagocytosis of tumor cells expressing cell surface CRT, by dendritic cells. CRT is also important to the success of certain chemotherapies [10–12]. In addition, CRT is integrally important in the process of healing cutaneous wounds [13, 14]. These functions are directed by the surface-exposed or secreted protein form of CRT [14, 15]. The translocation of CRT to the cell surface can be induced by ER stress in some cell types, which is triggered by various stimuli including anthracyclines, irradiation and reduction of ER Ca2+ levels [16–18]. Importantly, since exogenous CRT rescues CRT-deficient cells in numerous and different CRT-dependent functions, such as adhesion, migration, phagocytosis, and immunoregulation , exogenously supplied CRT has significant therapeutic potential for a variety of indications including impaired diabetic wound healing and cancer therapy [8, 10, 11, 13, 14, 19]. In fact, chronic wound healing and particularly impaired cutaneous healing as a consequence of diabetes is a global serious unmet medical need and economic challenge (26 million worldwide patients). Taken together, 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.
Here we show that expression of a full-length human CRT precursor in yeast cells can be employed to obtain a high level of secretion of mature native recombinant CRT protein. Fed-batch fermentation of P. pastoris culture resulted in more than 1.5 g/l CRT secretion yield. Comparison of yeast-secreted recombinant CRT to native CRT isolated from human placenta showed that the recombinant protein has apparent similar molecular and functional properties as the native protein. From this study, we can conclude that yeast is an excellent host for industrially relevant production of native recombinant CRT protein.
Expression and purification of human CRT from yeasts S. cerevisiae and P. pastoris from culture medium
Recombinant human CRT secreted by yeast and CRT protein isolated from human placenta have similar molecular features
Oligomerization and conformation of yeast-secreted and placental CRTs
Oligomerization analysis by native and blue native PAGE showed that both placental and recombinant CRTs were present only in monomeric form (Fig. 2). However, we observed a slight difference in mobility of CRTs purified from human placenta and yeast culture media. Native human protein migrated slightly faster than recombinant CRTs, and this suggested a conformational difference. In order to check possible influence of different buffers used for storage of recombinant and placental CRTs, we have transferred the latter to storage buffer of recombinant CRTs and repeated both native PAGEs. Indeed, buffer change resulted in slower migration of placental CRT and it became very similar to that of yeast-secreted proteins (Additional file 5).
Functional analysis of the recombinant and native human CRTs
Using the scratch plate method as an in vitro wound healing assay, after 24 h of treatment, 75–95 % wound closure was achieved with all CRT proteins with a peak concentration of 1.0 ng/ml compared to the 0.5 % FBS untreated control, which showed 53 % closure at 24 h (Fig. 7a). Specifically, at this peak concentration, a statistically significant induction of wound closure by native CRT of 81.3 % (p ≤ 0.05) and CRT produced by P. pastoris of 94.6 % (p ≤ 0.001) over the untreated control was obtained (n = 1). Whereas statistical significance in induction of wound closure at this concentration was not reached by CRT from S. cerevisiae, there was no difference among the three CRTs at 1.0 ng/ml. To test the sensitivity of CRT responsiveness in stimulating migration as measures by wound closure on the scratch, the plates were analyzed at 6 h post-treatment. As shown in Fig. 7b, there was a statistically significant induction of wound closure for native and S. cerevisiae-derived CRTs at both 1.0 and 10 ng/ml (n = 2). At the peak concentration of 10 ng/ml CRT, native and S. cerevisiae-secreted CRTs stimulated wound closure at 9.3 % (p ≤ 0.01) and 8.7 % (p ≤ 0.05), respectively over the untreated control of 5.5 % closure. There weren’t any statistically significant differences among all CRTs at any dose. Notably, the peak of activity in induction of wound closure was similar with native CRT compared to the two yeast-derived recombinant CRTs suggesting that the functional integrity of yeast-derived CRT was preserved. As described above, the downward trend in activity at 100 ng/ml was obtained previously [13, 14] and likely represents receptor saturation at higher concentrations. Whereas receptor signaling via the LRP1 has been shown for migration , which might be operating in the scratch plate assay, the receptor that mediates proliferation is unknown. Earlier we reported that ATPase activity of yeast-secreted human GRP78/BiP protein exceeded the activity of E. coli-derived recombinant human BiP threefold , whereas yeast-secreted human ERp57 catalyzed the reduction of insulin at a faster rate than analogous recombinant human protein expressed in E. coli . However, comparative biological activities to native human proteins were precluded since native protein was not available. The use of native CRT protein isolated from human placenta herein afforded the opportunity to optimally compare the yeast-secreted recombinant protein with purified native human CRT. Similar functional activity of native CRT and recombinant CRT strongly suggest that the synthesized and secreted recombinant CRT should have the same effect on wound healing in vivo, as cellular migration and proliferation are critical functions that enable the wound repair process . As animal studies showed that topical CRT has the potential to be used as an effective therapeutic agent for chronic wounds, including poor healing diabetic wounds , the molecular identity between native and yeast-derived CRTs underscores the utility of recombinant CRT secreted from yeast as having clinical and commercial value for wound healing. As the protein consists of the same amino acid sequence, CRT should not be immunogenic as a side effect of treatment.
High-level secretion of recombinant human CRT by fed-batch fermentation of P. pastoris
Development of biopharmaceuticals requires the biosynthesis process to be fully controlled and industrially relevant. P. pastoris is known as a highly efficient expression system with well-developed high culture density fermentation protocols. Therefore, we performed synthesis of recombinant secreted human CRT using cultivation of P. pastoris in a bioreactor and show the results, which can be adapted for the production of human CRT protein at an industrial scale.
Secretion efficiency of human CRT in yeast
Significance and applicability of the results
With a growing demand for human CRT biotherapeutics, we introduce yeast S. cerevisiae and P. pastoris as excellent hosts for easily purified, high-level production of human CRT. Here we demonstrated that the native signal sequence of human CRT precursor is correctly cleaved and directs secretion in yeast cells. High-level secretion of human CRT using fed-batch fermentation of high-density P. pastoris culture in a bioreactor allowed efficient production of secreted CRT protein in grams per liter scale. To the best of our knowledge, this is one of the highest secretion levels achieved using native human protein signal sequences in yeast. Comparable results after optimization were achieved only with human serum albumin (HSA) . Our data suggest that molecular integrity and functional activity of yeast-secreted recombinant CRT is identical to native CRT protein from human placenta. From these results, we can extrapolate that CRT secreted by yeast should be inherently biologically active in any functional in vitro and in vivo acitivity (harboured by native CRT) with applicability to the development of new biopharmaceuticals such as has been shown for normal and diabetic wound healing [13, 14]. Moreover, it was recently shown that this same yeast-secreted CRT can also be used as a cancer treatment adjuvant in combination with photodynamic therapy . Yeast-secreted recombinant protein may be advantageous in such applications, because it corresponds to the native analogue insofar as possible. Furthermore, yeast-derived heterologous proteins are free of toxic contaminations and are excellent tools for developing biopharmaceuticals, because S. cerevisiae is acknowledged as a GRAS (generally regarded as safe) organism. Therefore, secretory expression of native recombinant human CRT in yeast could be exploited for efficient and safe production of potential therapeutic agents.
Our results show that yeast is an excellent host for efficient and industrial scale production of native recombinant human CRT. Yeast cells are able to recognize and correctly process the native signal sequence of human CRT precursor. Yeast secretes mature CRT protein into the culture medium, instead of retaining the protein in the ER. Unusual high-level secretion enabled CRT production in grams per liter scale with a simple and cost-effective purification scheme. Recombinant CRT secreted by yeast appears seemingly identical to the native protein from human placenta with respect to its molecular integrity and functional activity in vitro. The yeast expression and purification systems for human CRT offer a new way of producing large quantities of human protein for therapeutic application.
Plasmids, yeast strains, transformation and selection of transformants
DNA manipulations were performed according to standard procedures , bacterial recombinants were screened in Escherichia coli DH5αF′ cells. A cDNA encoding full-length human calreticulin precursor (GenBank Acc. no. M84739) was amplified from a commercial human adult liver cDNA library (Clontech) by PCR using specific oligonucleotide primers CRTF (gta tct aga aca atg ctg cta tcc gtg ccg ttg) and CRTR (cag tct aga cta cag ctc gtc ctt ggc ctg), digested with restriction endonuclease (RE) XbaI (recognition sites in sequences of primers are indicated in bold) and cloned into yeast expression vectors pFDC  and pPIC3.5K (Invitrogen) into RE sites XbaI and AvrII under the control of S. cerevisiae PGK1 or P. pastoris AOX1 promoters, respectively. Cloned CRT gene coding sequence (beginning from start codon ATG and ending with STOP codon TAG) was verified by DNA sequencing and generated plasmids pFDC-CRT and pPIC3.5K-CRT were used for transformation of yeast S. cerevisiae strain AH22 (MATa leu2 his4) and P. pastoris strain GS115 (his4), respectively. S. cerevisiae transformants were selected by resistance to formaldehyde  and harboured multicopy autonomously replicating plasmid pFDC-CRT, whereas multicopy P. pastoris transformants were selected by resistance to G418  and Mut+ clone CPp9 with the most efficient secretion of CRT protein was chosen for further experiments. Both yeasts were used for expression of the full length CRT protein precursor including the native N-terminal signal peptide.
Protein expression in shake-flask cultivations and purification of recombinant CRT from yeast culture media
Saccharomyces cerevisiae cells harbouring plasmid pFDC-CRT were grown for 36 h in YEPD (yeast extract 1 %, peptone 2 %, dextrose 2 %) medium. P. pastoris transformants with multicopy integrations of expression vector pPIC3.5K-CRT were initially grown in Ygly medium containing glycerol as carbon source (yeast extract 1 %, peptone 2 %, glycerol 1 %, biotin 2 × 10−5 %) for 24 h up to OD of 18–20, and culture medium was changed to Ymet containing methanol (yeast extract 1 %, peptone 2 %, methanol 1 %, biotin 2 × 10−5 %) for induction of CRT cDNA expression. Then flasks were further incubated in the shaker at 30 °C, 220 rpm for 120 h. 1 % of methanol was added every 8 h to maintain protein expression. Both P. pastoris and S. cerevisiae cultures were centrifuged at 10,000×g for 10 min at 4 °C. Supernatants were collected and stored on ice. Protocol for CRT purification from culture media was the same for both yeasts.
Yeast culture medium was microfiltered through 1.6 µM (Sartorius Stedim Biotech, cat. no. FT-3-1101-047), 0.45 µM (Sartorius Stedim Biotech, cat. no. 15406-47) and 0.2 µM (Sartorius Stedim Biotech, cat. no. 15407-47-MIN) filters using a Pressure Filter Holder (Sartorius Stedim Biotech, cat. no. 16249) and a vacuum pump. After microfiltration, proteins from the culture medium were concentrated and transferred into the binding buffer (20 mM l-histidine, 100 mM NaCl, pH 5.5) through tangential ultrafiltration using cassettes with 100 kDa cut-off membranes (Sartorius Stedim Biotech, cat. no. VF20P4) and a peristaltic pump. Further, proteins were loaded onto the column packed with Q Sepharose FastFlow resin (GE Healthcare, cat. no. 17-0510-10) equilibrated in the same buffer. The column was washed with 5 volumes of binding buffer and bound proteins were eluted with a NaCl concentration gradient (100–500 mM). CRT was eluted in a single peak with approximately 250 mM NaCl. Elution fractions containing purified recombinant protein were pooled and buffer was exchanged to CRT storage buffer (20 mM Tris–HCl, 150 mM NaCl, 3 mM CaCl2, pH 7.5) using Sephadex G25 column. Protein was stored frozen at −70 °C.
Purification of native CRT from human placenta
Human placentas were donated for research with written consent from women who had given birth to healthy children at Rigshospitalet, Copenhagen. At the Statens Serum Institut they were used anonymously for purification of CRT. Human placental CRT was purified as described previously . Briefly, a placenta was homogenized twice in 20 mM BisTris, 1 mM CaCl2, pH 7.5 with protease inhibitors (Complete) and with intermittent centrifugations. The residue was then extracted twice in 20 mM BisTris, 1 mM CaCl2, pH 7.5, 1 % Triton X-114 to release ER luminal proteins. Membrane proteins were removed by phase separation at 37 °C and large proteins removed from the water phase by ammonium sulphate precipitation (337 g/l) overnight at 5 °C. The supernatant was ultradiafiltrated against 20 mM Tris, 1 mM CaCl2, pH 7.5 and chromatographed on a Q Sepharose column equilibrated in the same buffer and eluted with a linear NaCl gradient. CRT-containing fractions were pooled and concentrated to 1 mg/ml in 20 mM Tris/HCl, pH 7.5 using a Centriprep filter. Purified placental CRT was stored frozen at −20 °C.
Partial proteolysis of CRT with trypsin in the presence of calcium
Partial digestion of CRT with trypsin was performed according to previously described procedures [26, 31]. Purified yeast-secreted CRT was diluted to 1 mg/ml concentration in storage buffer containing 3 mM CaCl2. To avoid influence of different storage buffers, native placental CRT was also transferred to storage buffer with 3 mM CaCl2 as for recombinant CRTs, and diluted to the same concentration. Digestion was performed at 37 °C in 50 µl volume by adding 1 μl of 0.5 mg/ml trypsin [ratio of CRT:trypsin was 100:1 (w/w)]. In a control tube, calcium was removed by adding EGTA to 5 mM concentration. The reaction was stopped after 60 min, by adding 1 mM PMSF. The samples were boiled, loaded onto the gels, and SDS-PAGE was performed.
Function of yeast-derived calreticulin: cellular migration and proliferation assays
Cell proliferation An in vitro assay for cellular proliferation was performed as previously described [13, 14]. Primary low passage human dermal foreskin fibroblasts (CCD 1070SK; American Type Culture Collection, Manassas, VA, USA), grown in complete Eagle’s minimal essential medium (MEM; Gibco/Life Technologies, Carlsbad, CA, USA) containing 10 % fetal bovine serum (FBS), 2 mM glutamine (Mediatech, Manassas, VA, USA), 1.0 mM sodium pyruvate (Gibco) and antibiotics, were seeded at 2 × 103 cells per well in 96 well plates and grown to 50 % confluency (24 h). Subsequently, the cells were synchronized in media containing 0.5 % FBS for 24 h and treated with increasing concentrations of CRT (0–100 ng/ml) from placenta, or S. cerevisiae or P. pastoris for 72 h. CRT protein concentrations were determined by microBCA assay to standardize that equal amounts of each CRT were used. The assay was performed in triplicate for each test parameter. Media with 0.5 % FBS and 10 % FBS served as a negative and positive controls, respectively. Proliferation was determined by an MTS assay (CellTiter96 assay, G3580, Promega, Madison, WI, USA). Cells were incubated in MTS solution for 1 h and absorbance was measured at 490 nm (BioRad ELISA reader). The data are expressed as fold proliferation over untreated control (n = 6 experiments).
Cell migration The effect of native human placental and yeast-derived human CRT on cellular migration was assessed by the classic in vitro wound healing scratch plate assay, as described [13, 14]. Human dermal fibroblasts in complete MEM containing 10 % FBS were seeded at 2 × 104 cells per well in 24 well plates and grown to 70–80 % confluency. The cells were switched to media containing 0.5 % FBS for 24 h, and then wounds were created down the center of each well by scratching with a 200 µl pipette tip, and the loose cells removed by washing with PBS. The edges of the wounds were denoted with a fine-tipped marker on the underside of the plate. The cells were treated for 6 or 24 h with increasing concentrations of CRT protein in 0.5 % FBS media. 0.5 and 10 % FBS served as negative and positive controls, respectively. Migration was terminated by incubating the cells with 0.025 % Coomassie blue in 10 % acetic acid, 45 % methanol for 10 min followed by washing twice with PBS. The plates were viewed under an inverted light microscope (Axiovert s-100; Zeiss, Thornwood, NY, USA) and images captured and cell migration calculated by measuring the area (pixels) not occupied by cells (i.e., open area remaining) to the area of the original scratch at time 0 using Metamorph software version 188.8.131.52 (Molecular Probes, Eugene OR, USA). The plates were normalized for cell density at a part of the plate distal to the scratch. The data are expressed as per cent wound closure (0 time compared to 6 or 24 h later) and the averages for each test parameter subjected to analysis of variance (ANOVA). Statistical significance for both proliferation and wound closure assays were performed using GraphPad Prism software (version 6). A two-way ANOVA followed by a Dunnett post hoc test by comparing each treatment value with 0.5 % FBS as the untreated control; statistical significance was (p < 0.05). The experiments were performed in triplicate (n = 2 experiments at 6 h and n = 1 at 24 h).
High cell density fed-batch fermentation of recombinant P. pastoris
For this study, selected Mut+ phenotype P. pastoris GS115 transformant strain CPp9 carrying multiple copies of human CRT gene under the control of AOX1 promoter, was used. A 200 ml culture for inoculation of bioreactor was prepared in two stages as described previously . First, a 20-ml primary seed YEPD medium was prepared using 200 μl of the user seed lot which was grown at 28 °C in an orbital shaker (New Brunswick Innova 40R) at 220 rpm for 24 h. This primary seed medium was used to inoculate a 200 ml secondary seed medium, containing fermentation media, to 0.1 OD that was grown for 16–18 h to 4–6 OD under the same conditions, to serve as inoculum for the bioreactor culture. Cultures were grown in baffled flasks whose volumes were 5× the culture volume to permit adequate aeration.
High-cell density fed-batch cultivation was performed as described previously  with several exceptions. Cultivation was carried out in a 5 l BIOSTAT-A plus (Sartorius Stedim Biotech) bioreactor interfaced with MFCS/DA software for data acquisition and control. A 200 ml inoculum, prepared as described above, was transferred to the bioreactor containing 1.8 l fermentation media. The fermentation media contained per liter: glycerol, 63 g; potassium sulfate, 14.67 g; calcium sulfate, 0.9 g; magnesium sulfate hepta-hydrate, 11.67 g; trace metal solution (PTM1), 4 ml; ammonium sulfate, 9 g; hexametaphosphate, 25 g. The PTM1 solution contained per liter: sodium iodide, 0.08 g; manganese sulfate mono-hydrate, 3.0 g; sodium molybdate di-hydrate, 0.2 g; boric acid, 0.02 g; zinc chloride, 20.0 g; ferric sulfate hepta-hydrate, 65.0 g; cupric sulfate penta-hydrate, 6.0 g; biotin, 0.2 g; and sulfuric acid, 5.0 ml. 0.2 ml of antifoam (Antifoam 204, SigmaAldrich) per 1 l media was added manually before inoculation to control foaming in the bioreactor. Aeration rate of 1 vvm was constant throughout the whole process. The dissolved oxygen (DO) throughout the whole process was controlled at 30 % saturation using an automatic DO control by agitation cascade between 410 and 750 rpm and oxygen supplementation. When a maximum 750 rpm was reached, pure oxygen was supplied through a gas blender to control dissolved oxygen at 30 % saturation.
Temperature during glycerol batch phase was maintained at 28 °C and pH at 5.0 with 28 % (v/v) NH4OH. After consumption of glycerol, indicated by an increase of the DO concentration, glycerol-fed batch phase was initiated. Temperature during glycerol-fed batch phase was maintained at 28 °C and pH at 7.0. 50 % glycerol (v/v) with PTM1 was fed at 20 ml/l/h for 1 h before being ramped down to 0 ml/l/h at a uniform rate over a 3-h period. The ramping down of glycerol marked the beginning of the transition phase and 2 ml/l of methanol was added to the bioreactor to allow the cells to adjust to methanol. Temperature during transition phase was ramped down to 20 °C and pH was maintained at 7.0. After transition phase methanol-fed batch phase (production of recombinant CRT) was initiated by the addition of methanol at a 2 ml/l/h rate which was increased to 3.8 ml/l/h at a uniform rate over a 6 h period and 3.8 ml/l/h methanol feed rate was maintained during whole methanol-fed batch. Temperature during methanol-fed batch phase was maintained at 20 °C and pH at 7.0. For DCW determination, cell pellets were washed once with distilled water and incubated in an open tube to a constant weight at 80 °C.
Tryptic peptide mass fingerprinting was carried out at the Proteomics Center in the Institute of Biochemistry of Vilnius University (Lithuania). S. cerevisiae-secreted CRT was analyzed by MALDI-TOF/TOF tandem MS/MS (mass spectrometry) as described previously . Trypsin digestion of P. pastoris-secreted CRT was done according to a modified FASP protocol as described by Wisniewski et al. . The nano-LC was coupled online with an HDMS Synapt G2 mass spectrometer (Waters Corporation, UK). LC–MS data were collected using data independent acquisition (DIA) mode MSE in combination with online ion mobility separation.
N-terminal sequencing of yeast-secreted human CRT protein by Edman degradation was performed by AltaBioscience.
The molecular masses of proteins were measured by ESI–MS using an Agilent Q-TOF 6520 mass spectrometer.
Protein concentrations were determined by Roti-Nanoquant Protein-assay (Carl Roth Gmbh., cat. no. K880) using BSA for calibration curve.
Densitometric analysis of SDS-PAGE gels scanned with ImageScanner III (GE Healthcare) was performed with ImageQuant TL (GE Healthcare) software using default settings. For quantitative analysis of secreted CRT, the culture medium was separated from cells by centrifugation, directly mixed with equal amount of 2× SDS-PAGE sample buffer , boiled and loaded onto gel lanes. Amounts of secreted CRT in culture medium were determined by comparison to a range of known amounts of BSA (Fermentas product #B14 was used) in a linear dynamic range (R2 > 0.99) of Coomassie-stained protein bands in SDS-PAA gels. To determine CRT purity in crude yeast culture media, total secreted yeast proteins were concentrated by precipitation and yeast protein impurities were evaluated by SDS-PAGE.
Precipitation of proteins from yeast culture medium for SDS-PAGE analysis was performed based on a defined methanol-chloroform-water mixture, as described earlier .
Mouse monoclonal anti-CRT antibody [FMC 75] was purchased from Abcam (cat. no. ab22683-100).
EČ conceived of the study, performed work on recombinant CRT expression and purification from yeast, high-density yeast fermentation, trypsin digestion and electrophoresis experiments, collected and analysed data, and revised manuscript. EŽ was involved in high-density yeast fermentation and trypsin digestion experiments. LIG and SCP performed CRT functional assays, JD analysed data and performed statistical analysis, and LIG revised manuscript. PH, GH purified native CRT from human placenta, were involved in native PAGE experiments, and revised the manuscript. MM and WAW were involved in functional CRT assays, MM analysed data, reviewed and revised the manuscript. RS helped to design the study, was involved in recombinant CRT expression and MS experiments, analysed, interpreted data, and drafted the manuscript. All authors read and approved the final manuscript.
The work of EČ, EŽ and RS was supported by the European Social Fund under the Global Grant measure grant no. VP1-3.1-ŠMM-07-K-02-038 (to R.S.). The study was also supported by grants from the Canadian Institutes of Health Research (CIHR) (to M.M.). W-A.W. is supported by a Frederick Banting and Charles Best Canada Graduate Doctoral Scholarship from CIHR. We are grateful to Audronė Rukšėnaitė (Vilnius University, Insitute of Biotechnology) and Dr. Algirdas Kaupinis (Vilnius University, Insitute of Biochemistry) for performing MS experiments. Kirsten Beth Hansen and Dorthe Tange Olsen are thanked for excellent technical assistance.
A part of the work presented in this paper was filed for international patent application WO/2014/011723.
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