Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants

Abstract

Asialoerythropoietin (asialo-EPO) is a desialylated form of human glycoprotein hormone erythropoietin (EPO), which has been reported to be neuro-, cardio-, and renoprotective in animal models of organ injuries. Since the current method of production of asialo-EPO from mammalian cell-made recombinant human EPO (rhuEPO^M) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPO^P). Despite achieving high expression levels in plants, its purification from plant extracts has remained a greater challenge, which has prevented studying its tissue-protective effects and translating it into clinical practice. In this study, a procedure was developed to purify asialo-rhuEPO^P from transgenic tobacco leaf tissues in two steps: ion-exchange chromatography based on its high pI (8.75) to separate it from acidic plant proteins, and immunoaffinity chromatography to obtain pure asialo-rhuEPO^P. Using this process, up to 31% of the asialo-rhuEPO^P could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPO^P expressed in tobacco plants could be purified in high yield and purity using minimal steps, which might be suitable for scale-up. Furthermore, the ion-exchange chromatography step together with the use of protein-specific antibody column could be used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues.

1. Introduction

Erythropoietin (EPO), a glyco-hormone, is best known for its regulatory role in the production of red blood cells. Recently, preclinical studies have shown that EPO and its derivatives display remarkable anti-apoptotic and broad tissue-protective effects against damage triggered by ischemia-reperfusion, or cytotoxic agents in the brain, the heart, the kidneys and the liver [1-4]. Unfortunately, the therapeutic application of mammalian cell-produced recombinant human EPO (rhuEPO^M) for cytoprotection was tempered by observations that it greatly increases thrombotic events and decreases survival rates because of its hematopoietic activity [5-8]. Moreover, the cytoprotective doses of EPO are much higher than those required for stimulation of erythropoiesis and its hematopoietic activity at these high doses can stimulate mass production of red blood cells causing more damage [9]. Therefore, cytoprotective EPO derivatives lacking hematopoietic activity are desired for cytoprotective purposes.

Asialoerythropoietin (asialo-EPO) is a desialylated form of EPO and non-erythropoietic. It is cytoprotective in animal models of stroke, nerve injury, spinal cord compression and ischemia-reperfusion injury of heart and kidneys [10-13], but has no adverse effects that are commonly associated with rhuEPO^M because of its hematopoietic activity. Asialo-EPO has also been shown to exhibit excellent protective effects in animal models of diabetes [14], diabetic nephropathy [15], wound healing [16] and autoimmune encephalomyelitis [17]. Despite encouraging results in many preclinical studies, it has found little or no use in clinical practice because of high cost involved its production by enzymatic desialylation of rhuEPO^M, which is very expensive (∼US$ 4,000 /mg) [18]. Thus, alternative methods to produce asialo-EPO inexpensively were highly desired.

Plants have emerged as a potential alternative to mammalian-based expression system. Plants are suited for asialoglycoprotein production because they lack sialylating capacity [19,20], but can synthesize complex N-glycans similar to mammalian N-glycans [21]. In addition to this, plant-based expression systems are cost effective, easy to scale up in production, and free of human pathogens [22]. Recently, we [23,24] and others [25-27] used plant-based expression to produce recombinant human asialo-EPO (asialo-rhuEPO^P). Despite achieving high expression levels of asialo-rhuEPO^P in plants, recovery and purification from transgenic plant tissues has remained a major challenge.

Downstream processing is an important part of biopharming, and can represent up to 80% of overall production costs [28,29]. Therefore, development of efficient extraction and purification processes is essential for favorable economics. In recent years, many strategies have been employed for efficient recovery and purification of a variety of recombinant proteins from plant tissues [28,29]. In many cases, it is required to develop specific processing steps for different protein, specific production host, and the required level of purity [28]. Affinity purification methods, wherein the protein of interest is appended with suitable affinity tag, and enriched by virtue of its specific binding properties to an immobilized ligand, have been evaluated for purification of asialo-rhuEPO^P from transgenic plant leaf tissues [24,26,30,31]. The Strep II tag, an eight amino acids long peptide (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), which exhibits high affinity toward streptavidin, was C-terminally fused to the EPO polypeptide chain [24,26,31]. Surprisingly, in all three studies, the Strep II tagged EPO failed to bind to the Strep-Tactin column. Jez et al (2013) [31] suggested that Strep II tag is either removed by proteolysis in tobacco plants or it is not accessible to bind to Strep-Tactin column. In another study, Castilho et al. (2013) [30] reported that the IgG-Fc domain fused to the C-terminus of EPO polypeptide chain was also cleaved off in tobacco plants. These results suggest that the C-terminus of EPO is highly susceptible to proteolysis when expressed in plants. The N-terminus of EPO is not selected because it contains a 27 amino acid long signal peptide that is also removed during protein maturation. Clearly, the method involving building a fusion partner or an affinity tag to EPO polypeptide chain is not suitable to facilitate purification of asialo-rhuEPO^P from transgenic leaf tissues. Purification procedures for isolation of tagless sialylated rhuEPO from mammalian cell cultures have been reported [32,33], but these are not suitable for purification of asialo-rhuEPO^P because it is a basic protein in contrast to sialylated rhuEPO, which is acidic in nature. Therefore, purification method for the isolation of asialo-rhuEPO^P from transgenic plant leaf tissues is urgently needed.

Recently, we purified asialo-rhuEPO^P from tobacco leaf tissues using preliminary fractionation with ammonium sulfate followed by immunoaffinity chromatography [24]. The yield of asialo-rhuEPO^P was however, extremely low (3-5%) and the lifetime of immunoaffinity column was shorter due to fouling of the column by plant extracts. In this study, we developed a two-step approach for the purification of asialo-rhuEPO^P from transgenic leaf tissues. The process involves extraction and recovery of asialo-rhuEPO^P into crude aqueous extracts followed by a purification process that includes a cation ion exchange chromatographic step to separate basic asialo-rhuEPO^P from abundant acidic ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO) and other acidic plant proteins, and an immunoaffinity chromatography step to obtain pure asialo-rhuEPO^P. Using this process, asialo-rhuEPO^P in a yield of 31% was obtained.

2. Materials and Methods

2.1. Ion exchange chromatography

Leaf tissues from high asialo-rhuEPO^P expressing transgenic tobacco line A56-5 [24] grown in a greenhouse was used for protein purification. Cation exchange chromatography was performed on SP-sepharose fast flow (FF) (GE Healthcare Biosciences, Pittsburg, PA, USA) column. An isoelectric point (pI) based method described by Adhikari et al. (2010) [34] was adopted to identify the optimal pH to bind asialo-rhuEPO^P to the SP-sepharose FF resin. Briefly, five grams of leaf tissues grounded into fine powder in liquid nitrogen were extracted with buffers shown in Table 1 whose pHs were 1 to 3.75 units below the theoretical pI of asialo-rhuEPO (ca pI 8.75). The above buffers contained 1 mM EDTA, 2% polyvinylpolyprrolidone and plant protease inhibitor cocktail (Sigma, Saint Louis, MO, USA). Extraction buffer was added to the samples in the ratio of 3 ml buffer to 1 g leaf tissues. The extracts were first passed through a double-thickness miracloth (EMD Millipore, Bellerica, MA, USA) followed by centrifugation at 20,000 g for 15 min to remove insoluble plant debris. The centrifugation step was repeated once in order to remove the remaining insolubles. The pH of the extracts was measured once again, and adjusted to the desired values if required. Soluble protein and asialo-rhuEPO^P in crude extracts were determined by Bradford method (1976) [35] and sandwich ELISA [24], respectively. The extracts, each containing about 2000 ng of asialo-rhuEPO^P were then applied to SP-sepharose FF column (1 × 5 cm), which had been equilibrated with buffers (10 CV) shown in Table 1. After washing with 5 column volumes (CV) of equilibration buffers, bound proteins were eluted using a step gradient of NaCl (0.1-0.5 M) in equilibration buffers. Protein was monitored by measuring absorption at A₂₈₀ nm while the amount of asialo-rhuEPO^P in each fraction was determined using sandwich ELISA as described previously [23].

Table 1.

Purification of asialo-rhuEPO^P using SP-sepharose FF column at different pHs.

Buffer pHa	Total asialo-rhuEPOPb (ng)	Recovered asialo-rhuEPOP (ng)	Yieldc (%)
5.0	2000	1219	60
5.75	2000	304	15
6.75	2000	180	9
7.75	2000	180	9

^aThe buffering compounds used to prepare buffers of different pH values were 50 mM sodium acetate for pH 5.0, 50 mM phosphate-citrate for pH 5.75, 50 mM MOPS for pH 6.75 and 50 mMTris-HCl for pH 7.75.

^bAsialo-rhuEPO^P was determined by sandwich ELISA.

^cThe % yield was calculated as (recovered asialo-rhuEPO^P/total asialo-rhuEPO^P) × 100%.

After establishing the optimal pH for binding and recovery from SP-sepharose FF column, the purification was scaled-up from 5 to 100 g. Briefly, leaf tissues (100 g) was extracted with 50 mM acetate buffer, pH 5 containing 1 mM EDTA, 2% polyvinylpolyprrolidone and plant protease inhibitor cocktail. Extract was applied to a pre-equilibrated SP-sepharose FF column (3 × 10 cm) at a flow rate of 2 ml/min. After washing with 10 CV of sodium acetate buffer (pH 5), bound proteins were eluted first with 0.3 M NaCl in 50 mM sodium acetate buffer, followed by 0.6 M and 1 M NaCl in the same buffer. Seven fractions of 20 ml each were collected and neutralized immediately with 1 M Tris-HCl (pH 8.8). Proteins and asialo-rhuEPO^P in each fraction were determined as described above.

2.2. Immunoaffinity chromatography

Immunoaffinity resin was prepared as described previously [23]. Briefly, 3 mg of rabbit polyclonal anti-EPO antibody (Sigma-Aldrich, Saint Louis, USA) containing 0.1% BSA in citrate-carbonate buffer, pH 10 was incubated with 6 ml of agarose resin overnight at 4 °C. After incubation, the resin was reduced with sodium cyanoborohydride, and the remaining active sites were blocked with 1 M Tris-HCl, pH 7.4. The resin was then washed with PBS containing 1 M NaCl, and stored in PBS containing 0.05% sodium azide at 4 °C until use. Asialo-rhuEPO^P fractions from SP-sepharose FF column were pooled (both peak 1 and 2 fractions), introduced into a 3.5 kD MWCO dialysis membrane, and the membrane was placed in a container containing PEG 12000 to concentrate the SP-sepharose fractions. The pH of the concentrate was then adjusted to pH 7.3 and mixed with BSA (0.5% w/v). Binding was performed by incubating the concentrated asialo-rhuEPO^P fractions with anti-EPO antibody-coupled resin overnight at 4 °C with shaking using an end-to-end rocking shaker. After binding, the antibody-coupled resin was packed into a column (1 × 10 cm) and washed with PBS to remove unbound proteins. Bound asialo-rhuEPO^P was eluted with 0.1 M glycine-HCl buffer, pH 2.5, neutralized immediately with 1 M Tris-HCl, pH 8.8; and asialo-rhuEPO^P in each fraction was determined using a sandwich ELISA as described previously [23].

2.3. SDS-PAGE and Western blotting

Purified asialo-rhuEPO^P was analyzed by SDS-PAGE under reducing conditions. Electrophoresis was performed according to the method of Laemmli (1970) [36] on a slab gel consisting of 12.5% acrylamide resolving gel and a 5% stacking gel. After protein separation, the gel was stained with coomassie brilliant blue. Western blotting was performed as described previously [23].

3. Results and Discussion

3.1. Ion-exchange chromatography

In contrast to sialylated rhuEPO, asialo-rhuEPO is a basic protein with theoretical pI of 8.75. It was reported recently by Adhikari et al. (2010) [34] that basic human proteins expressed in E. coli could be purified in a single step by cation-exchange chromatography if the pH of the binding buffer is merely one unit below the pI of basic recombinant proteins. Considering the facts that asialo-rhuEPO^P is a basic protein, and that the overall nature of native tobacco proteins including RuBisCO are acidic [37], we chose a strong cation exchanger SP-sepharose FF column to separate asialo-rhuEPO^P from RuBisCO and other acidic tobacco proteins. Since the theoretical pI of asialo-rhuEPO is 8.75, we chose extraction buffers whose pHs were 1, 2, 3 and 3.75 units below the theoretical pI of asialo-rhuEPO^P to investigate the optimal pH for purification of asialo-rhuEPO^P using SP-sepharose FF column. Because we observed that asialo-rhuEPO^P precipitated along with plant proteins at pH 4.75 (4 units below the theoretical pI), buffer of pH 5.0 was used as the lowest pH buffer for testing instead of 4.75.

The results of binding of asialo-rhuEPO^P to the SP-sepharose FF column at different pHs is summarized in Table 1. As can be seen, at pH 7.75 and 6.75 (1 and 2 unit below the theoretical pI of asialo-rhuEPO), no appreciable binding of asialo-rhuEPO^P to the SP-sepharose FF column occurred when crude protein extracts containing 2 μg of asialo-rhuEPO^P were applied. Lowering the pH more than 2 units however, increased the yield of asialo-rhuEPO^P. At pH 5 (3.75 units below the theoretical pI), about 60% of the asialo-rhuEPO^P was recovered. The results of binding and recovery of asialo-rhuEPO^P from SP-sepharose FF column are not in agreement with the results reported by Adhikari et al (2010) [34]. The discrepancy could be due to the presence of abundant soluble tobacco proteins at pHs 6.75 and 7.75, including RuBisCO in plant extracts [37], as well as chlorophylls, polysaccharides and secondary metabolites. These may interfere with binding of asialo-rhuEPO^P to the SP-sepharose column. Recently, Buyel and Fischer (2014) [38] reported that about 65% of the host cell proteins (HCP), including RuBisCO are precipitated at acidic pH (pH 4.5), and minimal binding of HCP and RuBisCO occurred at lower pH. The observed improvement in the yield of asialo-rhuEPO^P at pH 5.0 is very likely due to lesser interference from plant proteins and some other contaminants. The asialo-rhuEPO^P separated into two peaks on SP-sepharose FF column at all the pHs investigated (Fig. 1A, B, C and D). At pH 5, the major asialo-rhuEPO^P peak (peak I) eluted between 0.3-0.4 M NaCl whereas the minor peak (peak II) eluted between 0.4-0.5 M (Fig. 1A).

Fig. 1.

Purification of asialo-rhuEPO^P using SP-sepharose FF column at different pHs. (a) Sodium acetate buffer, pH 5; (b) phosphate-citrate buffer, pH 5.75; (c) MOPS, pH 6.75 and (d) Tris-HCl, pH 7.75. Extracts made by extracting 5 g of transgenic tobacco line A56-5 with different pH buffers were applied onto a SP-sepharose FF column (1 × 5 cm) and eluted with stepwise increases in the concentration of NaCl (0.1 to 0.5 M) as indicated (solid red line). Protein peaks (-□-) detected by absorbance at 280 nm, and the amount of asialo-rhuEPO^P (-■-) in each fraction measured by sandwich ELISA were plotted against fraction number.

Since higher yield of asialo-rhuEPO^P was obtained at pH 5 (sodium acetate buffer), we used this buffer and scaled up purification from 5 to 100 g. Leaf extracts were applied and eluted from SP-sepharose FF column as described in “Material and Methods”. Fig. 2A shows the elution profile of asialo-rhuEPO^P from SP-sepharose FF column. As observed earlier, asialo-rhuEPO^P separated into two peaks, a major peak eluting at 0.3 M NaCl and a minor peak at 0.6 M NaCl, implying protein charge heterogeneity. We used 0.6 M NaCl instead of 0.5 M to ensure complete elution of peak II. Western blot analysis showed that both peaks contained same three EPO glycoforms (26-30 kDa, Fig. 2B), suggesting that they bear same type of N-glycan chains, and that the N-glycosylation may not be a source of charge heterogeneity. The protein charge heterogeneity in asialo-rhuEPO^P could be due to protein aggregation or polypeptide chain modifications, such as deamidation or amino acid truncation [39]. This however, requires further investigation. After ion exchange chromatography, ∼2.0-fold purification was achieved and about 53% of the initial asialo-rhuEPO^P was recovered (Table 2).

Fig. 2.

Elution profile of asialo-rhuEPO^P from SP-sepharose FF column (A) and Western blot of asialo-rhuEPO^P peaks eluted from SP-sepharose FF column (B). A, Extracts of transgenic tobacco line A56-5 expressing asialo-rhuEPO were applied onto a SP-sepharose FF column (3 × 10 cm) and eluted by increases in the concentration of NaCl as indicated (solid red line). Fractions (20 ml each) were collected at a flow rate of 2 ml/min. Protein peaks (-□-) detected by absorbance at 280 nm, and the amount of asialo-rhuEPO^P (-■-) in each fraction measured by sandwich ELISA were plotted against fraction number. B, Asialo-rhuEPO^P peak 1 and peak 2 (25 and 50 μl of fraction number 4 and 10, respectively) were analyzed by Western blotting. Asialo-rhuEPO^P was detected as three major glycoforms of sizes 26, 28 and 30 kDa in both the peaks.

Table 2.

Purification of asialo-rhuEPO^P from transgenic tobacco leaf tissues.

Steps	Volume (ml)	Total proteina (mg)	Total asialo-rhuEPOP(μg)	Total activityb (IUb)	Specific activity (IU/mg)	Purification fold (x)c	Yield (%)d
Crude extract	260	178	29	3625	20	1	100
SP-sepharose	160	54	15.5	1937	36	1.8	53
IAC	24	0.02	9.0	1125	56250	2812	31

^aProtein concentration was determined by Bradford method.

^bInternational units (IU) of activity were computed by dividing nanograms of asialo-rhuEPO by 8 as described by Erbayraktar et al. (2003) [13]. One IU of erythropoietic activity is equal to 8 ng of asialo-rhuEPO.

^cThe purification fold was calculated based on the specific activity increased after each purification step comparing to that in initial crude extracts.

^dThe % yield in each purification step was calculated based on the total asialo-rhuEPO^P remained comparing to the initial amount of asialo-rhuEPO^P in crude extract.

3.2. Immunoaffinity chromatography

Since the asialo-rhuEPO^P fractions from SP-sepharose FF column still contained a large proportion of plant proteins (basic plant proteins such as extensins), an immunoaffinity chromatographic step was introduced to further purify asialo-rhuEPO^P. Previously, we tested three different anti-EPO antibodies immobilized on agarose support for the purification of asialo-rhuEPO^P and discovered that a polyclonal anti-EPO antibody produced in rabbit binds to asialo-rhuEPO^P efficiently [23]. In the current study, a 6 ml immunoaffinity column based on polyclonal anti-EPO antibody was used. Concentrated asialo-rhuEPO^P fraction rom SP-sepharose FF column was incubated with the anti-EPO antibody resin, and bound asialo-rhuEPO^P was eluted with 0.1 M glycine-HCl buffer, pH 2.5. Asialo-rhuEPO^P eluted as a single peak from immunoaffinity column (results not shown). Immunoaffinity chromatography resulted in an additional 1562-fold purification with the final yield of 31% (Table 2). This correspondence to a 6-fold improvement over our previous yield (3-5%) obtained by preliminary fractionation with ammonium sulfate followed by immunoaffinity chromatography [24]. Furthermore, adjusting the pH of the extracts to 5.0 and introducing the cation exchange chromatography step prevented the fouling of immunoaffinity column, and extended its lifetime by removing chlorophylls, alkaloids, and other unique host molecules that are present in the plant extracts [28]. The EPO binding capacity of immunoaffinity column remained unchanged even after more than five rounds of purification.

3.3. SDS-PAGE and Western blot analysis

SDS-PAGE analysis of purified asialo-rhuEPO^P showed four bands in the molecular weight range of 20-30 kDa, and a ∼60 kDa band corresponding to BSA fragment (BSA appears to be slightly degraded), which was added before loading to the immunoaffinity column to stabilize asialo-rhuEPO^P (Fig. 3A). Western blotting revealed three dominant immunoreactive bands of sizes 26, 28 and 30 kDa, and a weaker 20 kD band (Fig. 3B), indicating these to be asialo-rhuEPO glycoforms as previously reported [24].

Fig. 3.

SDS-PAGE (A) and Western blot (B) analyses of purified asialo-rhuEPO^P. (A) Five microgram each of purified asialo-rhuEPO^P and rhuEPO^M were separated on a 12.5% SDS-PAGE gel and stained with coommassie blue. Lane 1, pre-stained markers; lane 2, rhuEPO^M; lane 3, purified asialo-rhuEPO^P. (B) Ten nanogram each of asialo-rhuEPO^P and rhuEPO^M were used. After protein transfer, the blot was probed with mouse monoclonal anti-EPO antibody (1:1000 dilution), followed by peroxidase coupled anti-mouse secondary antibody (1:6000 dilution). Lane 1, rhuEPO^M; lane 2, asialo-rhuEPO^P.

4. Conclusion

In conclusion, we have demonstrated here that asialo-rhuEPO^P can be effectively purified from transgenic tobacco leaf tissues with high yield and purity in two steps after initial extraction. The ion exchange chromatography step served as an effective step for the removal of RuBisCO and other tobacco acidic proteins and the enrichment of the target protein. The cation exchange chromatography step can be potentially used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues. The immunoaffinity chromatography step effectively separated asialo-rhuEPO^P from remaining tobacco proteins. Approximately 31% of the initial asialo-rhuEPO^P could be recovered after the final step in purified form as judged by ELISA and shown by a coomassie stained SDS-PAGE. All of the steps incorporated in the purification process have the potential to be scaled up for large-scale protein production.