High-yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions
Sanjeewa Singhabahu1 John George2 ∗ David Bringloe2
1 Human
Genetics Unit, Faculty of Medicine, University of Colombo, Kynsey Road, Colombo 8, Sri Lanka
2 School
of Health, Sport and Bioscience, University of East London, Water Lane, London, UK
Abstract Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing
apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L−1 , a 336-fold increase over ADA produced by cell C 2014 suspensions transformed with a cytosolic construct. International Union of Biochemistry and Molecular Biology, Inc. Volume 00, Number 00, Pages 1–7, 2014
Keywords: adenosine deaminase, apoplast, molecular pharming, plant cell culture, recombinant protein, tobacco
1. Introduction In humans the conversion of metabolites adenosine and deoxyadenosine to inosine and deoxyinosine is catalyzed by adenosine deaminase or ADA (adenosine aminohydrolase, EC 3.5.4.4). In the absence of this enzyme these metabolites reach toxic levels in the plasma of individuals suffering from ADA deficiency, a form of inherited severe combined immunodeficiency
Abbreviations: ADA, adenosine deaminase; CaMV, cauliflower mosaic virus; ERT, enzyme replacement therapy; hGM-CSF, human-granulocyte macrophage colony-stimulating factor; HRGPs, hydroxyproline-rich glycoproteins; ORF, open reading frame; PEG, polyethylene glycol; RAmy3D, rice α-amylase 3D; SCID, severe combined immunodeficiency; TSP, total soluble protein. ∗ Address for correspondence: David Bringloe, PhD, School of Health, Sport and Bioscience, University of East London, Water Lane, London E15 4LZ, UK. Tel.: +44 (0)20 8223 4113; Fax: +44 (0)20 8223 4965; e-mail: [email protected]. Received 9 December 2013; accepted 3 May 2014 DOI: 10.1002/bab.1240
Published online in Wiley Online Library (wileyonlinelibrary.com)
(SCID). The toxic metabolites are taken up by lymphoid progenitors, causing impairment of T-cell development and hence immunodeficiency. ADA deficiency treatments are based on restoration of a functional immune system and can be attained by three main management options that include bone marrow transplant, somatic gene therapy, and enzyme replacement therapy (ERT) [1]. Because the first two options are not always available or successful, the third option, ERT with exogenously supplied ADA, is the most common treatment and has been used for more than 20 years to treat ADA-deficient patients [1]. ERT supplies exogenous bovine ADA through twice-weekly intramuscular injections of a polyethylene glycol conjugate (PEGADA). Normally, patients who are less than 1 year old when they start receiving this therapy respond well with an improved immune system and can expect a good quality of life [2, 3]. It would seem that when other options are unavailable ERT with PEG-ADA provides a life-saving therapy with sustained clinical benefits for at least a decade if treatment is continued beyond 6 months. Long-term ERT seems to be well tolerated, and most patients in therapy remain free of opportunistic or abnormally frequent infections [3]. Complications to this treatment have however arisen, where some patients develop neutralizing
1
Biotechnology and Applied Biochemistry antibodies to the bovine enzyme conjugate. These individuals therefore may well benefit from ADA administered as the human rather than the bovine form. Furthermore, as patients age, more frequent applications of the drug may also be necessary to keep the toxic metabolites at an acceptable level. The cost of the treatment, however, is prohibitively high, standing at $200,000–$300,000 per patient annually, and production of a recombinant human ADA in mammalian systems would likely incur further production costs, making more frequent administration of the drug impractical [2]. A cheaper alternative source of a recombinant form of human ADA would therefore be expedient. The expression of functional, recombinant human ADA in whole tobacco plants, with a view to large-scale production, has previously been reported [4]. However, using plant cell suspension cultures to express foreign proteins encompasses some advantages over whole-plant systems [5]. A major advantage is the direction of recombinant proteins to the apoplast through subcellular targeting: an approach used to protect proteins from proteolytic degradation in the cytosol and also eject such proteins into the medium of cell suspensions, where they can be more readily purified. Signal sequences from proteins, such as tobacco pathogenesis-related protein (PR1a), when used in combination with recombinant proteins, can target foreign proteins into the apoplast [6]. Another extensively studied N-terminal apoplast-directing signal is found in plant extensins, a well-characterized hydroxyproline-rich glycoprotein (HRGP) family, which form a major component of the cell wall [7]. Like the PR1a signal peptide, incorporation of an extensin signal peptide at the N-terminus of recombinant proteins directs secretion into the apoplast. Using the extensin signal peptide Francisco et al. [8] directed bryodin-1, a potent ribosome-inactivating protein, into the culture medium. Successful apoplast direction of recombinant therapeutic proteins has also been reported by Xu et al. [9], where an extensin signal peptide was used in conjunction with a C-terminal hydroxyproline (Hyp)-rich peptide to produce a Hyp-rich glycoprotein. Chimeric expression of such glycoproteins, where a short Cterminal Hyp-Ser sequence directs extensive O-glycosylation, leads to the addition of arabinooligosaccharides. Using these apoplast-directed conjugates Xu et al. [9] significantly increased yields of recombinant interferon-α2-arabinogalactan peptides by as much as 350–1,400-fold. With increasing demand for therapeutic proteins, plant cell culture systems have been developed as an alternative system for producing therapeutically imperative proteins. Recently, a recombinant protein, human glucocerebrosidase (taliglucerase alfa), which is used in ERT to treat Gaucher’s disease, has been produced by Protalix Biotherapeutics. This enzyme was the first plant-cell-produced human enzyme to be approved by the US Food and Drug Administration in 2012 [10]. Although there are many reports of recombinant proteins produced in plant cell suspension culture systems, using tomato, rice, soybean, and tobacco plants [11], tobacco BY-2 cell suspensions are the most commonly used cell system.
2
Plant cell cultures hold the potential to be an alternative production system for pharmaceutical proteins; however, a lesser amount of recombinant protein yield is a major limitation in commercializing this system. Hence, a wide array of strategies have been employed, in an attempt to enhance protein yield at the molecular, cell culture, and downstreamprocessing steps. Various molecular strategies have been widely used in recent years to enhance the yield of foreign proteins in plant cell cultures [12]. These include approaches such as enhancement of both gene transcription and translational efficiencies and incorporation of both novel protein-fusion and secretory-pathway-targeting technologies. In this study all of the above technologies were employed in an attempt to increase the expression of recombinant ADA in transformed BY-2 calli and cell suspensions. The resulting transgenic calli and cell suspensions were assessed for ADA enzyme activities and ADA protein integrity.
2. Materials and Methods 2.1. Construction of ADA expression vectors A human ADA cDNA with no modifications that contained the native 5 and 3 untranslated regions (UTRs) was inserted into a pGREEN0029 vector under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator (pcDNA˙ADA; Fig. 1a), as described by Singhabahu et al. [4]. A second construct, p5 -PR1a˙ADA (Fig. 1b), was made from a PCR-amplified ADA cDNA open reading frame (ORF) inserted into pGREEN0029 and modified at the 5 end with an 80-bp tobacco mosaic virus omega () UTR translational enhancer sequence and a 90-bp PR1a apoplast targeting signal sequence [13]. A third construct, p5 -Ext˙ADA (Fig. 1c), was made from a PCR-amplified ADA cDNA ORF inserted into pGREENI0029, modified at the 5 end with an 80-bp tobacco mosaic virus UTR sequence and a 78-bp tobacco extensin signal sequence [8]. A fourth construct, p5 -Ext˙ADA-3 HP (Fig. 1d), was made from a PCR-amplified ADA cDNA ORF construct inserted into pGREENI0029, modified at the 5 end with both an 80-bp tobacco mosaic virus UTR sequence and a 78-bp tobacco extensin signal sequence. It was also modified at the 3 end with a 63-bp serine–hydroxyproline sequence, which was designed to incorporate 20 repeating serine–hydroxyproline amino acid residues at the C-terminus of the recombinant ADA [9]. In addition, a thrombin cleavage site was inserted between the last codon of the ADA ORF and the extra hydroxyproline codons.
2.2. BY-2 tobacco cell transformation and growth of calli and cell suspensions Agrobacteria transformed with the constructs mentioned earlier were used to transform tobacco BY-2 cell cultures, using a method described by Rempel and Nelson [14]. Transformed calli were visible after 20–30 days and transferred onto new mannitol salt (MS) agar plates, containing appropriate amounts of carbenicillin, cefotaxime, and kanamycin, and were grown
High-Yield Production of ADA in BY-2 Suspensions
FIG. 1
Schematic diagram of the gene constructs: (a) pcDNA_ADA, (b) p5 -PR1a_ADA, (c) p5 -Ext_ADA, (d) p5 -Ext_ADA-3 HP.
for a further 7–14 days, in the dark, at 25 ◦ C, prior to expression analysis. To initiate suspension cultures a small part (2 g) of transformed Nicotiana tabacum BY-2 calli were cut into small 2 mm2 pieces and transferred into 20 mL of fresh sterile MS culture medium in a 100-mL conical flask. The cultures were shaken at 120 rpm in the dark, at 25 ◦ C, for 7 days, after which the tobacco BY-2 starter suspension was transferred into 100 mL of fresh sterile MS culture medium in a 250-mL conical flask. Tobacco BY-2 cell suspensions were propagated weekly by inoculating 100 mL of MS medium with 20 mL of a one-week-old suspension culture.
Biotechnology and Applied Biochemistry
2.3. Protein extraction, Western blot analysis, and quantitation of expressed ADA Tissue samples (100 mg) taken from transformed cells in suspension or solid calli were ground to a fine powder in liquid nitrogen by using an Eppendorf tube grinder in an extraction buffer containing 50 mM potassium phosphate, pH 7.5, and 15 mg polyvinylpyrrolidone (PVP). Plant extract supernatants were collected after centrifugation at 10,000g for 10 Min. Total soluble protein (TSP) content of the tissue sample was determined using Bradford Reagent (Bio-Rad, Hemel Hempstead, UK). Protein samples containing 2 mg of protein were denatured at 95 ◦ C for 5 Min in SDS-PAGE sample buffer and subsequently electrophoresed on a 12% SDS-polyacrylamide gel. Prior to this the BY-2 calli extract of the ADA-hyroxyproline fusion protein was mixed with 20 units of thrombin and
3
Biotechnology and Applied Biochemistry
FIG. 2
ADA-specific activities measured in BY-2 calli transformed with various cytosolic and apoplast-directing constructs. Each circle represents ADA activity from an independent transformant, and the yellow diamond denotes the median value. The number of calli assayed is shown in brackets after each construct.
incubated at 22 ◦ C for 16 H. Bovine ADA (Sigma-Aldrich, UK) (100 ng) was used as a positive control. Separated protein samples were transferred to a Hybond C nylon membrane (GE Healthcare, Amersham, UK) by a method based on that of Polvino et al. [15]. Blotted proteins were probed with a rabbit antibovine ADA polyclonal antibody used at a dilution of 1:3,000 and a horseradish-peroxidase-conjugated antibody as the secondary reagent (Novus Biologicals, Cambridge, UK). The immune complexes were detected with a Lumi-LightPLUS enhanced chemiluminesence kit (Roche Diagnostics, Burgess Hill, UK). TotalLab Quant software was used to analyze Western blots. The areas of hybridizing bands present on blots were compared with the areas of quantified control ADA bands to calculate approximate relative amounts of recombinant ADA protein in micrograms. The activity of plant-derived human ADA in protein-extracted samples was measured using a kit (Bioquant, Nashville, Tennessee, USA). Essentially, tobacco calli (100–200 mg) were ground to a fine powder in a microfuge tube, using a microfuge tube grinder in liquid nitrogen, and
4
homogenized in 400 μL of chilled extraction buffer containing 50 mM potassium phosphate, pH 7.5, supplemented with 37.5 g L−1 PVP, molecular weight 40,000. The homogenate was then subjected to centrifugation at 10,000g for 10 Min at 4 ◦ C to remove plant cell debris. From the soluble protein solution, 5 μL was removed; enzyme assays were set up according to the manufacturer’s instructions, and absorbance was monitored at 550 nm in a Thermo-Fisher Multiskan plate reader (ThermoFisher Scientific, Waltham, MA, USA) to obtain absorbance per-minute values. Assays were performed in triplicate, and the average rate of absorbance change (A Min−1 ) was calculated from three separate assays per transformant. ADA activity in units per liter protein extract was calculated and expressed as units of enzyme specific activity per milligram of TSP. Similarly, 5 μL of calf intestinal ADA (Sigma-Aldrich) was analyzed as a control.
3. Results and Discussion 3.1. ADA enzyme expression in transformed BY-2 calli To begin with, tobacco BY-2 cells in suspension were transformed with three different constructs containing apoplastdirecting signals (p5 -PR1a˙ADA, p5 -Ext˙ADA, p5 Ext˙ADA-3 HP) and one construct containing a native human cDNA (pcDNA˙ADA) (Fig. 1). The BY-2 calli lines transformed with ADA constructs were assessed for expression levels after
High-Yield Production of ADA in BY-2 Suspensions
FIG. 3
Western blot of proteins extracted from seven transgenic tobacco calli transformed with ADA constructs: lane 1, bovine ADA; lane 2, thrombin-treated p5 -Ext_ADA-3 HP calli 17; lane 3, 5 -Ext-ADA calli 26; lane 4, 5 -PR1a_ADA calli 5; lane 5, pcDNA_ADA calli 7; lane 6, thrombin-treated p5 -Ext_ADA-3 HP calli 8; lane 7, 5 -Ext-ADA calli 29; lane 8, 5 -PR1a_ADA calli 6; lane 9, non-transformed; and lane 10, Invitrogen Benchmark protein ladder.
5 to 6 weeks. The transformed calli were flash-frozen in liquid nitrogen and ground to a fine powder. TSPs were extracted, and the calli extracts were assayed for ADA enzyme activity, to determine ADA levels, and a Bradford assay was performed, to determine the total amount of soluble proteins. Up to 30 transgenic calli lines of different apoplast-directed constructs were analyzed as shown in Fig. 2. Data obtained from calli transformed with the native human ADA cDNA construct were used as a control (pcDNA˙ADA) with which to compare ADA levels in the corresponding calli transformed with the three apoplast-directed constructs. Any two constructs compared were combined and ranked by ascending expression levels, and their U values were calculated using a Mann–Whitney statistical test, as described by Pollard [16]. A pairwise comparison of ADA activities measured in the three apoplast-targeted constructs with ADA activities measured in calli transformed with the human cDNA construct showed that ADA levels are significantly different in all three targeted constructs. Furthermore, when the highest outlying ADA specific activities of these constructs are compared with the highest activity obtained from calli transformed with pcDNA˙ADA, an increase of 1.5-fold to 3.0-fold in ADA specific activity is observed. Moreover, 23 transgenic calli lines from the apoplast-targeted constructs exhibited a higher ADA specific activity than the highest-expressing pcDNA˙ADA construct (Fig. 2). Western blot analysis of the four cell lines transformed with each construct also indicated the integrity of the ADA protein, with a single band present, migrating at about 41 kDa for constructs pcDNA˙ADA, p5 -PR1a˙ADA, and p5 -Ext˙ADA (Fig. 3). The band present at about 41 kDa is comparable in size to a product observed previously when whole transgenic tobacco plants transformed with human ADA constructs were subjected to Western blot analysis [4]. The reported size of both human and bovine ADA is 41 kDa [17, 18]. The thrombintreated hydroxyproline-tagged p5 -Ext˙ADA-3 HP extract was
Biotechnology and Applied Biochemistry
also expected to run at about 40 kDa. Indeed a band present at about this molecular weight is apparent in Fig. 3, lanes 2 and 6. However, a broad, higher-molecular-weight band, migrating at about 70–80 kDa, was also observed in the same lanes. The presence of this band is thought to be due to incomplete thrombin digestion of the ADA hydroxyproline fusion protein, which, because of the inclusion of 20 extra amino acid residues, was predicted to be extensively glycosylated. The increase in size of the hydroxyproline-tagged ADA, by about 30–40 kDa, is similar to the increase in size, reported by Xu et al. [9], of the molecular mass of hydroxyproline-tagged α-interferon. Here the apparent size range of the higher-molecular-weight product present on Western blots was attributed to microheterogeneity in attached glycans. The observation that the lower-molecularweight product in Fig. 3, lanes 2 and 6, is a much more condensed band suggests that it is free of any attached glycans.
3.2. ADA enzyme expression in transformed BY-2 cell suspensions Undifferentiated plant calli can be separated and propagated in culture media in controlled conditions to generate cell suspension cultures. In this study, BY-2 cell suspension cultures, generated from calli transformed with various ADA cDNA constructs (Fig. 1), were investigated for extra- and intracellular ADA enzyme activities over a period of 15 days. The highest-expressing transformed BY-2 calli cell lines were selected (Fig. 2), and suspension cultures were generated in MS media supplemented with 0.75 g L−1 of PVP (molecular weight 360,000). In initial experiments PVP was shown to dramatically improve ADA enzyme activity when included in the culture medium as described by LaCount et al. [19]. PVP was therefore subsequently included in all experiments. Aliquots of BY-2 cell suspension cultures were collected on alternate days, starting from day 0 up to day 15. The collected samples were subjected to centrifugation to separate the cells from the culture medium, which was then tested for ADA enzyme activity by using an ADA assay to determine the extracellular enzyme activity secreted into the culture medium. Intracellular ADA activities were assayed at the same time from pelleted cells flash-frozen in liquid nitrogen. Eight samples taken over 15 days from BY-2 cell suspensions transformed with various constructs were analyzed, and ADA activities are shown in units per liter (Fig. 4a, b). A cytosolic construct, pCDNA˙ADA, containing human 5 and 3 UTRs was used as a basal construct with which to compare the corresponding ADA-apoplastdirected constructs. The highest extracellular ADA activity was measured on day 7 for the p5 -Ext˙ADA-3 HP transformed cell line, which measured 739 U L−1 , a 336-fold increase over the cytosolic construct (Table 1). Extracellular ADAactivity of the p5 Ext˙ADA-3 HP transformed cell line peaked on day 7, followed by a drastic decline in ADA activity in the next 2 days and increased ADA activity thereafter. In summary there is between a 30-fold and 336-fold increase in secreted ADA activity when apoplast-targeting constructs are compared with the cytosolic
5
Biotechnology and Applied Biochemistry
FIG. 4
(a) Extracellular ADA activities (U L−1 ); (b) intracellular ADA activities (U L−1 ); and (c) average TSP (g L−1 ) measured in four transformed BY-2 cell suspensions transformed with various cytosolic and apoplast-directing constructs.
control on day 7. This contrasts with between a 6.4-fold and 62-fold increase when comparing total activities of both intraand extracellular ADA and with between a 4.3-fold and 27fold increase when activities are normalized for TSP on day 7 (Table 1). In a similar experiment investigating recombinant monoclonal antibody levels in tobacco cell suspensions, Sharp and Doran [20] also observed an increase in recombinant protein levels in the medium on day 16 of sampling after a drastic reduction of protein levels in the suspension media
6
following peak production on day 7. A similar but less striking pattern of ADA activity was also observed in the construct p5 -PR1a˙ADA transformed cell line, whereas the ADA activity of the construct p5 -Ext˙ADA transformed cell line showed a constant level of ADA from day 3 to day 11, followed by a steady decrease between day 13 and day 15. As shown in Fig. 4a, the p5 -Ext˙ADA-3 HP transformed cell line showed a second large sudden increase of ADA levels on day 13 of sampling. The second peak in ADA activity is thought to be due to cell death and release of nutrients into the medium. It is postulated that cells stop dying on day 13 and begin a second phase of growth, as Fig. 4c shows an increase in cell suspension TSP on day 13. This second burst of growth with concomitant ADA production is thought to be triggered by nutrients released from dying cells. As shown in Fig. 4 the highest intracellular ADA activity in suspensions was measured in the cell line transformed with the construct p5 -Ext˙ADA-3 HP (19 U L−1 (or 0.4 mg L−1 ). By contrast, the highest extracellular ADA activity in the same line was 739 U L−1 (or 16 mg L−1 ), which is a 40-fold increase in the extracellular ADA levels as compared with the intracellular levels. The highest intracellular ADA activity of the p5 -Ext˙ADA transformed line measured 8.4 U L−1 (0.18 mg L−1 ), and the extracellular ADA activity measured 68.4 U L−1 (1.5 mg L−1 ). This represents an 8-fold increase in the extracellular ADA levels compared with the intracellular levels. These data suggest that most of the recombinant ADA targeted at the apoplast by N-terminal signal sequences had been secreted into the medium, leaving little intracellular ADA enzyme. It is also possible that additional, highly glycosylated hydroxyproline motifs serve to stabilize the recombinant ADA, eventually enhancing extracellular yields, in accordance with the findings of Xu et al. [9], where extensive glycosylation was also found to stabilize recombinant protein production. To corroborate this hypothesis, further investigations should be performed subjecting purified recombinant ADA to both protease and glycosidase digestion. Measurement of TSP in cell suspensions over time is an indicator of cell growth [21]. As shown in Fig. 4c, the amount of TSP measured in all four transformed tobacco BY-2 cell suspensions shows a rapid increase by up to 2-fold from day 0 to day 5, followed by an almost constant level of TSP until day 11. A reduction in TSP occurred on day 13 in all four cell lines, followed by a slight increase on day 15. Nagata et al. [22] and Horemans et al. [23] also demonstrated that the rapid growth of BY-2 cells in suspension occurs from day 0 to 5, followed by a stationary phase. In contrast, Sharp and Doran [20] reported that the growth of tobacco cells in suspension does not peak with foreign-protein production, where maximum secreted levels of recombinant monoclonal antibody were observed before maximum biomass was reached. Hence, higher amounts of biomass (or TSP) do not always peak with higher foreignprotein production. It may be that factors such as culture conditions and recombinant construct design determine when peak levels of targeted proteins are secreted into the medium.
High-Yield Production of ADA in BY-2 Suspensions
Intracellular, extracellular, and total ADA activities in transgenic tobacco BY-2 suspensions on day 7
TABLE 1 Construct
Intracellular (U L−1 )
p cDNA_ADA p5 -PR1a_ADA
9.9
2.3
Total ADA (U mg−1 TSP)
12.2
1.50×10−3
186
199.1
1.10 × 10−2
8.4
68.4
76.8
6.42 × 10−3
19.1
739.4
758.5
4.04 × 10−2
In agreement with previous attempts to express biopharmaceuticals in plant cell suspensions by using apoplast targeting signals, the transformed BY-2 cell suspensions of the current investigation showed considerably higher levels of secreted ADA. Approximate extracellular ADA levels of 16, 4, and 1.5 mg L−1 were observed respectively in BY-2 cell suspensions transformed with the p5 -Ext˙ADA-3 HP, p5 -PR1a˙ADA, and p5 -Ext˙ADA constructs. These are comparable to the levels of 30 mg L−1 measured for an extensin–bryodin construct [8] and 10 mg L−1 measured for a human α-iduronidase construct in tobacco cell suspensions [24], but lower than, for example, 129 mg L−1 of human-granulocyte macrophage colony-stimulating factor (hGM-CSF) driven by a rice α-amylase leader (RAmy3D) [25] and 247 mg L−1 of human α1-antitrypsin driven by a RAmy3D promoter measured in rice cell suspensions [26] Long-term ERT of ADA patients requires injections of 30 U kg−1 on a weekly basis [27]; so 16 mg ADA per liter (or 739 U L−1 ) produced by the best-performing BY-2 cell line is a reasonably successful start to make plant cell production a viable alternative source of recombinant ADA in the future. Any hydroxyproline tag associated with ADA produced in this cell line would, however, possibly need to be removed during the manufacturing process, adding an additional downstream cost to recombinant protein production.
4. Acknowledgements S. Singhabahu was funded by a scholarship from the University of East London.
5. References [1] Booth, C., Hershfield, M., Notarangelo, L., Buckley, R., Hoenig, M., Mahlaoui, N., Cavazzana-Calvo, M., Aiuti, A., and Gaspar, H.B. (2007) Clin. Immunol. 123(2), 139–147. [2] Chan, B., Wara, D., Bastian, J., Hershfield, M.S., Bohnsack, J., Azen, C.G., Parkman, R., Weinberg, K., and Kohn, D.B. (2005) Clin. Immunol. 117(2), 133–143.
Biotechnology and Applied Biochemistry
Total ADA (U L−1 )
13.1
p5 -Ext_ADA p5 -Ext_ADA-3 HP
Extracellular (U L−1 )
[3] Gaspar, H.B., Aiuti, A., Porta, F., Candotti, F., Hershfield, M.S., and Notarangelo, L.D. (2009) Blood 114(17), 3524–3532. [4] Singhabahu, S., George, J., and Bringloe, D.H. (2013) Transgenic Res. 22(3), 643–649. [5] Hellwig, S., Drossard, J., Twyman, R.M., and Fischer, R. (2004) Nat. Biotechnol. 22, 1415–1422. [6] Pen, J., van Ooyen, A.J.J., van den Elzena, P.J.M., Quaxb, W.J., and Hoekema, A. (1993) Indus. Crops Prod. Sci. 1, 241–250. [7] Showalter, A.M. (1993) Plant Cell 5, 9–23. [8] Francisco, J.A., Gawlak, A.L., Miller, M., Bathe, J., Russell, D., and Chace, D. (1997) Bioconjug. Chem. 8, 708–713. [9] Xu, J., Tan, L., Goodrum, K.J., and Kieliszewski, M.J. (2007) Biotechnol. Bioeng. 97(5), 997–1008. [10] Pfizer Press Releases. (2012) Accessed 2 May 2012 at: http:// www.pfizer.com/news/press_releases/. [11] Huang, T.K., and McDonald, K.A. (2009) Biochem. Eng. J. 45(3), 168–184. [12] Desai, P., Shrivastava, N., and Padh, H. (2010) Biotechnol. Adv. 28, 427–435. [13] Ziegler, T.M., Thomas, S.R., and Danna, K.J. (2000) Mol. Breed. 6, 37–46. [14] Rempel, H.C., and Nelson, L.M. (1995) Transgenic Res. 4, 199–207. [15] Polvino, W.J., Saravis, C.A., Sampson, C.E., and Cook, R.B. (1983) Electrophoresis 4(5), 368–369. [16] Pollard, J.H. (1977) A Handbook of Numerical and Statistical Techniques with Examples Mainly from the Life Sciences. pp.116–127, Cambridge University Press, Cambridge, UK. [17] Wiginton, D.A., Adrian, G.S., and Hutton, J.J. (1984) Nucl. Acids Res. 12(5), 2439–2446. [18] Kelly, M.A., Vestling, M.M., Murphy, C.M., Hua, S., Sumpter, T., and Fenselau, C. (1996) J. Pharm. Biomed. Anal. 14(11), 1513–1519. [19] LaCount, W., An, G., and Lee, J.M. (1997) Biotechnol. Lett. 19(1), 93–96. [20] Sharp, J.M., and Doran, P.M. (2001) Biotechnol. Prog. 17(6), 979–992. [21] Lee, S.Y., Hur, W., Cho, G.H., and Kim, D.I. (2001) Biotechnol. Bioprocess Eng. 6, 72–74 [22] Nagata, T., Nemoto, Y., and Hasezawa, S. (1992) Internat. Rev. Cytol. 132, 1–30. [23] Horemans, N., Potters, G., De Wilde, L., and Caubergs, R.J. (2003) Plant Physiol. 133(1), 361–367. [24] Fu, L. H., Miao, Y. S., Lo, S.W., Seto, T.C., Sun, S.S.M., Xu, Z.F., Clemens, S., Clarke, L. A., Kermode, A. R., and Jiang, L. (2009) Plant Sci. 177, 668–675. [25] Shin, Y.J., Hong, S.Y., Kwon, T.H., Jang, Y.S., and Yang, M.S. (2003) Biotechnol. Bioeng. 82(7), 778–783. [26] McDonald, K. A., Hong, L. M., Trombly, D. M., Xie, Q., and Jackman, A. P. (2005) Biotechnol. Prog. 21, 728–734. [27] Booth, C., and Gaspar, H.C. (2009) Biologic. Targets Ther. 3, 349–358.
7
Sanjeewa Singhabahu1 John George2 ∗ David Bringloe2
1 Human
Genetics Unit, Faculty of Medicine, University of Colombo, Kynsey Road, Colombo 8, Sri Lanka
2 School
of Health, Sport and Bioscience, University of East London, Water Lane, London, UK
Abstract Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing
apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L−1 , a 336-fold increase over ADA produced by cell C 2014 suspensions transformed with a cytosolic construct. International Union of Biochemistry and Molecular Biology, Inc. Volume 00, Number 00, Pages 1–7, 2014
Keywords: adenosine deaminase, apoplast, molecular pharming, plant cell culture, recombinant protein, tobacco
1. Introduction In humans the conversion of metabolites adenosine and deoxyadenosine to inosine and deoxyinosine is catalyzed by adenosine deaminase or ADA (adenosine aminohydrolase, EC 3.5.4.4). In the absence of this enzyme these metabolites reach toxic levels in the plasma of individuals suffering from ADA deficiency, a form of inherited severe combined immunodeficiency
Abbreviations: ADA, adenosine deaminase; CaMV, cauliflower mosaic virus; ERT, enzyme replacement therapy; hGM-CSF, human-granulocyte macrophage colony-stimulating factor; HRGPs, hydroxyproline-rich glycoproteins; ORF, open reading frame; PEG, polyethylene glycol; RAmy3D, rice α-amylase 3D; SCID, severe combined immunodeficiency; TSP, total soluble protein. ∗ Address for correspondence: David Bringloe, PhD, School of Health, Sport and Bioscience, University of East London, Water Lane, London E15 4LZ, UK. Tel.: +44 (0)20 8223 4113; Fax: +44 (0)20 8223 4965; e-mail: [email protected]. Received 9 December 2013; accepted 3 May 2014 DOI: 10.1002/bab.1240
Published online in Wiley Online Library (wileyonlinelibrary.com)
(SCID). The toxic metabolites are taken up by lymphoid progenitors, causing impairment of T-cell development and hence immunodeficiency. ADA deficiency treatments are based on restoration of a functional immune system and can be attained by three main management options that include bone marrow transplant, somatic gene therapy, and enzyme replacement therapy (ERT) [1]. Because the first two options are not always available or successful, the third option, ERT with exogenously supplied ADA, is the most common treatment and has been used for more than 20 years to treat ADA-deficient patients [1]. ERT supplies exogenous bovine ADA through twice-weekly intramuscular injections of a polyethylene glycol conjugate (PEGADA). Normally, patients who are less than 1 year old when they start receiving this therapy respond well with an improved immune system and can expect a good quality of life [2, 3]. It would seem that when other options are unavailable ERT with PEG-ADA provides a life-saving therapy with sustained clinical benefits for at least a decade if treatment is continued beyond 6 months. Long-term ERT seems to be well tolerated, and most patients in therapy remain free of opportunistic or abnormally frequent infections [3]. Complications to this treatment have however arisen, where some patients develop neutralizing
1
Biotechnology and Applied Biochemistry antibodies to the bovine enzyme conjugate. These individuals therefore may well benefit from ADA administered as the human rather than the bovine form. Furthermore, as patients age, more frequent applications of the drug may also be necessary to keep the toxic metabolites at an acceptable level. The cost of the treatment, however, is prohibitively high, standing at $200,000–$300,000 per patient annually, and production of a recombinant human ADA in mammalian systems would likely incur further production costs, making more frequent administration of the drug impractical [2]. A cheaper alternative source of a recombinant form of human ADA would therefore be expedient. The expression of functional, recombinant human ADA in whole tobacco plants, with a view to large-scale production, has previously been reported [4]. However, using plant cell suspension cultures to express foreign proteins encompasses some advantages over whole-plant systems [5]. A major advantage is the direction of recombinant proteins to the apoplast through subcellular targeting: an approach used to protect proteins from proteolytic degradation in the cytosol and also eject such proteins into the medium of cell suspensions, where they can be more readily purified. Signal sequences from proteins, such as tobacco pathogenesis-related protein (PR1a), when used in combination with recombinant proteins, can target foreign proteins into the apoplast [6]. Another extensively studied N-terminal apoplast-directing signal is found in plant extensins, a well-characterized hydroxyproline-rich glycoprotein (HRGP) family, which form a major component of the cell wall [7]. Like the PR1a signal peptide, incorporation of an extensin signal peptide at the N-terminus of recombinant proteins directs secretion into the apoplast. Using the extensin signal peptide Francisco et al. [8] directed bryodin-1, a potent ribosome-inactivating protein, into the culture medium. Successful apoplast direction of recombinant therapeutic proteins has also been reported by Xu et al. [9], where an extensin signal peptide was used in conjunction with a C-terminal hydroxyproline (Hyp)-rich peptide to produce a Hyp-rich glycoprotein. Chimeric expression of such glycoproteins, where a short Cterminal Hyp-Ser sequence directs extensive O-glycosylation, leads to the addition of arabinooligosaccharides. Using these apoplast-directed conjugates Xu et al. [9] significantly increased yields of recombinant interferon-α2-arabinogalactan peptides by as much as 350–1,400-fold. With increasing demand for therapeutic proteins, plant cell culture systems have been developed as an alternative system for producing therapeutically imperative proteins. Recently, a recombinant protein, human glucocerebrosidase (taliglucerase alfa), which is used in ERT to treat Gaucher’s disease, has been produced by Protalix Biotherapeutics. This enzyme was the first plant-cell-produced human enzyme to be approved by the US Food and Drug Administration in 2012 [10]. Although there are many reports of recombinant proteins produced in plant cell suspension culture systems, using tomato, rice, soybean, and tobacco plants [11], tobacco BY-2 cell suspensions are the most commonly used cell system.
2
Plant cell cultures hold the potential to be an alternative production system for pharmaceutical proteins; however, a lesser amount of recombinant protein yield is a major limitation in commercializing this system. Hence, a wide array of strategies have been employed, in an attempt to enhance protein yield at the molecular, cell culture, and downstreamprocessing steps. Various molecular strategies have been widely used in recent years to enhance the yield of foreign proteins in plant cell cultures [12]. These include approaches such as enhancement of both gene transcription and translational efficiencies and incorporation of both novel protein-fusion and secretory-pathway-targeting technologies. In this study all of the above technologies were employed in an attempt to increase the expression of recombinant ADA in transformed BY-2 calli and cell suspensions. The resulting transgenic calli and cell suspensions were assessed for ADA enzyme activities and ADA protein integrity.
2. Materials and Methods 2.1. Construction of ADA expression vectors A human ADA cDNA with no modifications that contained the native 5 and 3 untranslated regions (UTRs) was inserted into a pGREEN0029 vector under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator (pcDNA˙ADA; Fig. 1a), as described by Singhabahu et al. [4]. A second construct, p5 -PR1a˙ADA (Fig. 1b), was made from a PCR-amplified ADA cDNA open reading frame (ORF) inserted into pGREEN0029 and modified at the 5 end with an 80-bp tobacco mosaic virus omega () UTR translational enhancer sequence and a 90-bp PR1a apoplast targeting signal sequence [13]. A third construct, p5 -Ext˙ADA (Fig. 1c), was made from a PCR-amplified ADA cDNA ORF inserted into pGREENI0029, modified at the 5 end with an 80-bp tobacco mosaic virus UTR sequence and a 78-bp tobacco extensin signal sequence [8]. A fourth construct, p5 -Ext˙ADA-3 HP (Fig. 1d), was made from a PCR-amplified ADA cDNA ORF construct inserted into pGREENI0029, modified at the 5 end with both an 80-bp tobacco mosaic virus UTR sequence and a 78-bp tobacco extensin signal sequence. It was also modified at the 3 end with a 63-bp serine–hydroxyproline sequence, which was designed to incorporate 20 repeating serine–hydroxyproline amino acid residues at the C-terminus of the recombinant ADA [9]. In addition, a thrombin cleavage site was inserted between the last codon of the ADA ORF and the extra hydroxyproline codons.
2.2. BY-2 tobacco cell transformation and growth of calli and cell suspensions Agrobacteria transformed with the constructs mentioned earlier were used to transform tobacco BY-2 cell cultures, using a method described by Rempel and Nelson [14]. Transformed calli were visible after 20–30 days and transferred onto new mannitol salt (MS) agar plates, containing appropriate amounts of carbenicillin, cefotaxime, and kanamycin, and were grown
High-Yield Production of ADA in BY-2 Suspensions
FIG. 1
Schematic diagram of the gene constructs: (a) pcDNA_ADA, (b) p5 -PR1a_ADA, (c) p5 -Ext_ADA, (d) p5 -Ext_ADA-3 HP.
for a further 7–14 days, in the dark, at 25 ◦ C, prior to expression analysis. To initiate suspension cultures a small part (2 g) of transformed Nicotiana tabacum BY-2 calli were cut into small 2 mm2 pieces and transferred into 20 mL of fresh sterile MS culture medium in a 100-mL conical flask. The cultures were shaken at 120 rpm in the dark, at 25 ◦ C, for 7 days, after which the tobacco BY-2 starter suspension was transferred into 100 mL of fresh sterile MS culture medium in a 250-mL conical flask. Tobacco BY-2 cell suspensions were propagated weekly by inoculating 100 mL of MS medium with 20 mL of a one-week-old suspension culture.
Biotechnology and Applied Biochemistry
2.3. Protein extraction, Western blot analysis, and quantitation of expressed ADA Tissue samples (100 mg) taken from transformed cells in suspension or solid calli were ground to a fine powder in liquid nitrogen by using an Eppendorf tube grinder in an extraction buffer containing 50 mM potassium phosphate, pH 7.5, and 15 mg polyvinylpyrrolidone (PVP). Plant extract supernatants were collected after centrifugation at 10,000g for 10 Min. Total soluble protein (TSP) content of the tissue sample was determined using Bradford Reagent (Bio-Rad, Hemel Hempstead, UK). Protein samples containing 2 mg of protein were denatured at 95 ◦ C for 5 Min in SDS-PAGE sample buffer and subsequently electrophoresed on a 12% SDS-polyacrylamide gel. Prior to this the BY-2 calli extract of the ADA-hyroxyproline fusion protein was mixed with 20 units of thrombin and
3
Biotechnology and Applied Biochemistry
FIG. 2
ADA-specific activities measured in BY-2 calli transformed with various cytosolic and apoplast-directing constructs. Each circle represents ADA activity from an independent transformant, and the yellow diamond denotes the median value. The number of calli assayed is shown in brackets after each construct.
incubated at 22 ◦ C for 16 H. Bovine ADA (Sigma-Aldrich, UK) (100 ng) was used as a positive control. Separated protein samples were transferred to a Hybond C nylon membrane (GE Healthcare, Amersham, UK) by a method based on that of Polvino et al. [15]. Blotted proteins were probed with a rabbit antibovine ADA polyclonal antibody used at a dilution of 1:3,000 and a horseradish-peroxidase-conjugated antibody as the secondary reagent (Novus Biologicals, Cambridge, UK). The immune complexes were detected with a Lumi-LightPLUS enhanced chemiluminesence kit (Roche Diagnostics, Burgess Hill, UK). TotalLab Quant software was used to analyze Western blots. The areas of hybridizing bands present on blots were compared with the areas of quantified control ADA bands to calculate approximate relative amounts of recombinant ADA protein in micrograms. The activity of plant-derived human ADA in protein-extracted samples was measured using a kit (Bioquant, Nashville, Tennessee, USA). Essentially, tobacco calli (100–200 mg) were ground to a fine powder in a microfuge tube, using a microfuge tube grinder in liquid nitrogen, and
4
homogenized in 400 μL of chilled extraction buffer containing 50 mM potassium phosphate, pH 7.5, supplemented with 37.5 g L−1 PVP, molecular weight 40,000. The homogenate was then subjected to centrifugation at 10,000g for 10 Min at 4 ◦ C to remove plant cell debris. From the soluble protein solution, 5 μL was removed; enzyme assays were set up according to the manufacturer’s instructions, and absorbance was monitored at 550 nm in a Thermo-Fisher Multiskan plate reader (ThermoFisher Scientific, Waltham, MA, USA) to obtain absorbance per-minute values. Assays were performed in triplicate, and the average rate of absorbance change (A Min−1 ) was calculated from three separate assays per transformant. ADA activity in units per liter protein extract was calculated and expressed as units of enzyme specific activity per milligram of TSP. Similarly, 5 μL of calf intestinal ADA (Sigma-Aldrich) was analyzed as a control.
3. Results and Discussion 3.1. ADA enzyme expression in transformed BY-2 calli To begin with, tobacco BY-2 cells in suspension were transformed with three different constructs containing apoplastdirecting signals (p5 -PR1a˙ADA, p5 -Ext˙ADA, p5 Ext˙ADA-3 HP) and one construct containing a native human cDNA (pcDNA˙ADA) (Fig. 1). The BY-2 calli lines transformed with ADA constructs were assessed for expression levels after
High-Yield Production of ADA in BY-2 Suspensions
FIG. 3
Western blot of proteins extracted from seven transgenic tobacco calli transformed with ADA constructs: lane 1, bovine ADA; lane 2, thrombin-treated p5 -Ext_ADA-3 HP calli 17; lane 3, 5 -Ext-ADA calli 26; lane 4, 5 -PR1a_ADA calli 5; lane 5, pcDNA_ADA calli 7; lane 6, thrombin-treated p5 -Ext_ADA-3 HP calli 8; lane 7, 5 -Ext-ADA calli 29; lane 8, 5 -PR1a_ADA calli 6; lane 9, non-transformed; and lane 10, Invitrogen Benchmark protein ladder.
5 to 6 weeks. The transformed calli were flash-frozen in liquid nitrogen and ground to a fine powder. TSPs were extracted, and the calli extracts were assayed for ADA enzyme activity, to determine ADA levels, and a Bradford assay was performed, to determine the total amount of soluble proteins. Up to 30 transgenic calli lines of different apoplast-directed constructs were analyzed as shown in Fig. 2. Data obtained from calli transformed with the native human ADA cDNA construct were used as a control (pcDNA˙ADA) with which to compare ADA levels in the corresponding calli transformed with the three apoplast-directed constructs. Any two constructs compared were combined and ranked by ascending expression levels, and their U values were calculated using a Mann–Whitney statistical test, as described by Pollard [16]. A pairwise comparison of ADA activities measured in the three apoplast-targeted constructs with ADA activities measured in calli transformed with the human cDNA construct showed that ADA levels are significantly different in all three targeted constructs. Furthermore, when the highest outlying ADA specific activities of these constructs are compared with the highest activity obtained from calli transformed with pcDNA˙ADA, an increase of 1.5-fold to 3.0-fold in ADA specific activity is observed. Moreover, 23 transgenic calli lines from the apoplast-targeted constructs exhibited a higher ADA specific activity than the highest-expressing pcDNA˙ADA construct (Fig. 2). Western blot analysis of the four cell lines transformed with each construct also indicated the integrity of the ADA protein, with a single band present, migrating at about 41 kDa for constructs pcDNA˙ADA, p5 -PR1a˙ADA, and p5 -Ext˙ADA (Fig. 3). The band present at about 41 kDa is comparable in size to a product observed previously when whole transgenic tobacco plants transformed with human ADA constructs were subjected to Western blot analysis [4]. The reported size of both human and bovine ADA is 41 kDa [17, 18]. The thrombintreated hydroxyproline-tagged p5 -Ext˙ADA-3 HP extract was
Biotechnology and Applied Biochemistry
also expected to run at about 40 kDa. Indeed a band present at about this molecular weight is apparent in Fig. 3, lanes 2 and 6. However, a broad, higher-molecular-weight band, migrating at about 70–80 kDa, was also observed in the same lanes. The presence of this band is thought to be due to incomplete thrombin digestion of the ADA hydroxyproline fusion protein, which, because of the inclusion of 20 extra amino acid residues, was predicted to be extensively glycosylated. The increase in size of the hydroxyproline-tagged ADA, by about 30–40 kDa, is similar to the increase in size, reported by Xu et al. [9], of the molecular mass of hydroxyproline-tagged α-interferon. Here the apparent size range of the higher-molecular-weight product present on Western blots was attributed to microheterogeneity in attached glycans. The observation that the lower-molecularweight product in Fig. 3, lanes 2 and 6, is a much more condensed band suggests that it is free of any attached glycans.
3.2. ADA enzyme expression in transformed BY-2 cell suspensions Undifferentiated plant calli can be separated and propagated in culture media in controlled conditions to generate cell suspension cultures. In this study, BY-2 cell suspension cultures, generated from calli transformed with various ADA cDNA constructs (Fig. 1), were investigated for extra- and intracellular ADA enzyme activities over a period of 15 days. The highest-expressing transformed BY-2 calli cell lines were selected (Fig. 2), and suspension cultures were generated in MS media supplemented with 0.75 g L−1 of PVP (molecular weight 360,000). In initial experiments PVP was shown to dramatically improve ADA enzyme activity when included in the culture medium as described by LaCount et al. [19]. PVP was therefore subsequently included in all experiments. Aliquots of BY-2 cell suspension cultures were collected on alternate days, starting from day 0 up to day 15. The collected samples were subjected to centrifugation to separate the cells from the culture medium, which was then tested for ADA enzyme activity by using an ADA assay to determine the extracellular enzyme activity secreted into the culture medium. Intracellular ADA activities were assayed at the same time from pelleted cells flash-frozen in liquid nitrogen. Eight samples taken over 15 days from BY-2 cell suspensions transformed with various constructs were analyzed, and ADA activities are shown in units per liter (Fig. 4a, b). A cytosolic construct, pCDNA˙ADA, containing human 5 and 3 UTRs was used as a basal construct with which to compare the corresponding ADA-apoplastdirected constructs. The highest extracellular ADA activity was measured on day 7 for the p5 -Ext˙ADA-3 HP transformed cell line, which measured 739 U L−1 , a 336-fold increase over the cytosolic construct (Table 1). Extracellular ADAactivity of the p5 Ext˙ADA-3 HP transformed cell line peaked on day 7, followed by a drastic decline in ADA activity in the next 2 days and increased ADA activity thereafter. In summary there is between a 30-fold and 336-fold increase in secreted ADA activity when apoplast-targeting constructs are compared with the cytosolic
5
Biotechnology and Applied Biochemistry
FIG. 4
(a) Extracellular ADA activities (U L−1 ); (b) intracellular ADA activities (U L−1 ); and (c) average TSP (g L−1 ) measured in four transformed BY-2 cell suspensions transformed with various cytosolic and apoplast-directing constructs.
control on day 7. This contrasts with between a 6.4-fold and 62-fold increase when comparing total activities of both intraand extracellular ADA and with between a 4.3-fold and 27fold increase when activities are normalized for TSP on day 7 (Table 1). In a similar experiment investigating recombinant monoclonal antibody levels in tobacco cell suspensions, Sharp and Doran [20] also observed an increase in recombinant protein levels in the medium on day 16 of sampling after a drastic reduction of protein levels in the suspension media
6
following peak production on day 7. A similar but less striking pattern of ADA activity was also observed in the construct p5 -PR1a˙ADA transformed cell line, whereas the ADA activity of the construct p5 -Ext˙ADA transformed cell line showed a constant level of ADA from day 3 to day 11, followed by a steady decrease between day 13 and day 15. As shown in Fig. 4a, the p5 -Ext˙ADA-3 HP transformed cell line showed a second large sudden increase of ADA levels on day 13 of sampling. The second peak in ADA activity is thought to be due to cell death and release of nutrients into the medium. It is postulated that cells stop dying on day 13 and begin a second phase of growth, as Fig. 4c shows an increase in cell suspension TSP on day 13. This second burst of growth with concomitant ADA production is thought to be triggered by nutrients released from dying cells. As shown in Fig. 4 the highest intracellular ADA activity in suspensions was measured in the cell line transformed with the construct p5 -Ext˙ADA-3 HP (19 U L−1 (or 0.4 mg L−1 ). By contrast, the highest extracellular ADA activity in the same line was 739 U L−1 (or 16 mg L−1 ), which is a 40-fold increase in the extracellular ADA levels as compared with the intracellular levels. The highest intracellular ADA activity of the p5 -Ext˙ADA transformed line measured 8.4 U L−1 (0.18 mg L−1 ), and the extracellular ADA activity measured 68.4 U L−1 (1.5 mg L−1 ). This represents an 8-fold increase in the extracellular ADA levels compared with the intracellular levels. These data suggest that most of the recombinant ADA targeted at the apoplast by N-terminal signal sequences had been secreted into the medium, leaving little intracellular ADA enzyme. It is also possible that additional, highly glycosylated hydroxyproline motifs serve to stabilize the recombinant ADA, eventually enhancing extracellular yields, in accordance with the findings of Xu et al. [9], where extensive glycosylation was also found to stabilize recombinant protein production. To corroborate this hypothesis, further investigations should be performed subjecting purified recombinant ADA to both protease and glycosidase digestion. Measurement of TSP in cell suspensions over time is an indicator of cell growth [21]. As shown in Fig. 4c, the amount of TSP measured in all four transformed tobacco BY-2 cell suspensions shows a rapid increase by up to 2-fold from day 0 to day 5, followed by an almost constant level of TSP until day 11. A reduction in TSP occurred on day 13 in all four cell lines, followed by a slight increase on day 15. Nagata et al. [22] and Horemans et al. [23] also demonstrated that the rapid growth of BY-2 cells in suspension occurs from day 0 to 5, followed by a stationary phase. In contrast, Sharp and Doran [20] reported that the growth of tobacco cells in suspension does not peak with foreign-protein production, where maximum secreted levels of recombinant monoclonal antibody were observed before maximum biomass was reached. Hence, higher amounts of biomass (or TSP) do not always peak with higher foreignprotein production. It may be that factors such as culture conditions and recombinant construct design determine when peak levels of targeted proteins are secreted into the medium.
High-Yield Production of ADA in BY-2 Suspensions
Intracellular, extracellular, and total ADA activities in transgenic tobacco BY-2 suspensions on day 7
TABLE 1 Construct
Intracellular (U L−1 )
p cDNA_ADA p5 -PR1a_ADA
9.9
2.3
Total ADA (U mg−1 TSP)
12.2
1.50×10−3
186
199.1
1.10 × 10−2
8.4
68.4
76.8
6.42 × 10−3
19.1
739.4
758.5
4.04 × 10−2
In agreement with previous attempts to express biopharmaceuticals in plant cell suspensions by using apoplast targeting signals, the transformed BY-2 cell suspensions of the current investigation showed considerably higher levels of secreted ADA. Approximate extracellular ADA levels of 16, 4, and 1.5 mg L−1 were observed respectively in BY-2 cell suspensions transformed with the p5 -Ext˙ADA-3 HP, p5 -PR1a˙ADA, and p5 -Ext˙ADA constructs. These are comparable to the levels of 30 mg L−1 measured for an extensin–bryodin construct [8] and 10 mg L−1 measured for a human α-iduronidase construct in tobacco cell suspensions [24], but lower than, for example, 129 mg L−1 of human-granulocyte macrophage colony-stimulating factor (hGM-CSF) driven by a rice α-amylase leader (RAmy3D) [25] and 247 mg L−1 of human α1-antitrypsin driven by a RAmy3D promoter measured in rice cell suspensions [26] Long-term ERT of ADA patients requires injections of 30 U kg−1 on a weekly basis [27]; so 16 mg ADA per liter (or 739 U L−1 ) produced by the best-performing BY-2 cell line is a reasonably successful start to make plant cell production a viable alternative source of recombinant ADA in the future. Any hydroxyproline tag associated with ADA produced in this cell line would, however, possibly need to be removed during the manufacturing process, adding an additional downstream cost to recombinant protein production.
4. Acknowledgements S. Singhabahu was funded by a scholarship from the University of East London.
5. References [1] Booth, C., Hershfield, M., Notarangelo, L., Buckley, R., Hoenig, M., Mahlaoui, N., Cavazzana-Calvo, M., Aiuti, A., and Gaspar, H.B. (2007) Clin. Immunol. 123(2), 139–147. [2] Chan, B., Wara, D., Bastian, J., Hershfield, M.S., Bohnsack, J., Azen, C.G., Parkman, R., Weinberg, K., and Kohn, D.B. (2005) Clin. Immunol. 117(2), 133–143.
Biotechnology and Applied Biochemistry
Total ADA (U L−1 )
13.1
p5 -Ext_ADA p5 -Ext_ADA-3 HP
Extracellular (U L−1 )
[3] Gaspar, H.B., Aiuti, A., Porta, F., Candotti, F., Hershfield, M.S., and Notarangelo, L.D. (2009) Blood 114(17), 3524–3532. [4] Singhabahu, S., George, J., and Bringloe, D.H. (2013) Transgenic Res. 22(3), 643–649. [5] Hellwig, S., Drossard, J., Twyman, R.M., and Fischer, R. (2004) Nat. Biotechnol. 22, 1415–1422. [6] Pen, J., van Ooyen, A.J.J., van den Elzena, P.J.M., Quaxb, W.J., and Hoekema, A. (1993) Indus. Crops Prod. Sci. 1, 241–250. [7] Showalter, A.M. (1993) Plant Cell 5, 9–23. [8] Francisco, J.A., Gawlak, A.L., Miller, M., Bathe, J., Russell, D., and Chace, D. (1997) Bioconjug. Chem. 8, 708–713. [9] Xu, J., Tan, L., Goodrum, K.J., and Kieliszewski, M.J. (2007) Biotechnol. Bioeng. 97(5), 997–1008. [10] Pfizer Press Releases. (2012) Accessed 2 May 2012 at: http:// www.pfizer.com/news/press_releases/. [11] Huang, T.K., and McDonald, K.A. (2009) Biochem. Eng. J. 45(3), 168–184. [12] Desai, P., Shrivastava, N., and Padh, H. (2010) Biotechnol. Adv. 28, 427–435. [13] Ziegler, T.M., Thomas, S.R., and Danna, K.J. (2000) Mol. Breed. 6, 37–46. [14] Rempel, H.C., and Nelson, L.M. (1995) Transgenic Res. 4, 199–207. [15] Polvino, W.J., Saravis, C.A., Sampson, C.E., and Cook, R.B. (1983) Electrophoresis 4(5), 368–369. [16] Pollard, J.H. (1977) A Handbook of Numerical and Statistical Techniques with Examples Mainly from the Life Sciences. pp.116–127, Cambridge University Press, Cambridge, UK. [17] Wiginton, D.A., Adrian, G.S., and Hutton, J.J. (1984) Nucl. Acids Res. 12(5), 2439–2446. [18] Kelly, M.A., Vestling, M.M., Murphy, C.M., Hua, S., Sumpter, T., and Fenselau, C. (1996) J. Pharm. Biomed. Anal. 14(11), 1513–1519. [19] LaCount, W., An, G., and Lee, J.M. (1997) Biotechnol. Lett. 19(1), 93–96. [20] Sharp, J.M., and Doran, P.M. (2001) Biotechnol. Prog. 17(6), 979–992. [21] Lee, S.Y., Hur, W., Cho, G.H., and Kim, D.I. (2001) Biotechnol. Bioprocess Eng. 6, 72–74 [22] Nagata, T., Nemoto, Y., and Hasezawa, S. (1992) Internat. Rev. Cytol. 132, 1–30. [23] Horemans, N., Potters, G., De Wilde, L., and Caubergs, R.J. (2003) Plant Physiol. 133(1), 361–367. [24] Fu, L. H., Miao, Y. S., Lo, S.W., Seto, T.C., Sun, S.S.M., Xu, Z.F., Clemens, S., Clarke, L. A., Kermode, A. R., and Jiang, L. (2009) Plant Sci. 177, 668–675. [25] Shin, Y.J., Hong, S.Y., Kwon, T.H., Jang, Y.S., and Yang, M.S. (2003) Biotechnol. Bioeng. 82(7), 778–783. [26] McDonald, K. A., Hong, L. M., Trombly, D. M., Xie, Q., and Jackman, A. P. (2005) Biotechnol. Prog. 21, 728–734. [27] Booth, C., and Gaspar, H.C. (2009) Biologic. Targets Ther. 3, 349–358.
7
