- Email: [email protected]
Overexpression of PACEsol improves BMP-7 processing in recombinant CHO cells
Journal of Biotechnology 164 (2012) 336–339
Contents lists available at SciVerse ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Overexpression of PACEsol improves BMP-7 processing in recombinant CHO cells Madhavi Sathyamurthy a , Jae Seong Lee a , Jin Hyoung Park a , Yeon Jung Kim a , Ji Yeon Jeong b , Ju Woong Jang b , Gyun Min Lee a,∗ a b
Department of Biological Sciences and Graduate School of Nanoscience & Technology (WCU), KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea Institute of Biomaterial and Medical Engineering, Korea Bone Bank, 402 Gasan-dong, Geumcheon-gu, Seoul 153-782, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 September 2012 Received in revised form 28 December 2012 Accepted 16 January 2013 Available online 31 January 2013 Keywords: BMP-7 BMP-7 precursor CHO cells PACEsol
a b s t r a c t Bone morphogenetic protein-7 (BMP-7) is a member of the TGF- superfamily and plays a critical role in cartilage, bone, and kidney development. BMP-7 is synthesized as a large precursor and undergoes proteolytic cleavage by subtilisin-like proprotein convertase to secrete the functionally active mature dimer. When CHO cells producing recombinant human BMP-7 (rhBMP) (CHO-BMP-7) were cultivated in a serum-free suspension culture, a significant amount of unwanted precursor forms of rhBMP-7 (ca. 69% of total rhBMP-7), along with the mature form of rhBMP-7, was secreted into the culture medium, likely due to the insufficient amount of the proteolytic cleaving enzyme within the secretory pathway. In order to solve this problem, a soluble form of the paired basic amino acid cleaving enzyme (PACEsol), responsible for the majority of the processing events occurring in the constitutive secretory pathway in mammalian cells, was overexpressed in CHO-BMP-7 cells. Overexpression of PACEsol was effective in processing the precursor forms of BMP-7, while it did not significantly affect cell growth. As a result, the culture supernatants of CHO-BMP-7 cells overexpressing PACEsol contained almost 100% of the mature BMP-7 form. Taken together, the results show that PACEsol overexpression in CHO-BMP-7 cells is an efficient means of increasing the production of mature BMP-7 and facilitating the downstream purification steps by eliminating the need to remove the precursor forms. © 2013 Elsevier B.V. All rights reserved.
Bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor- (TGF-) superfamily, is a 35 kDa glycoprotein primarily known for its osteoinductive properties (Chen et al., 1995) and kidney development (Dudley et al., 1995; Gould et al., 2002). Recombinant human BMP-7 (rhBMP-7) has been developed as a potential therapeutic agent for the treatment of tibial nonunion and fusion of vertebral bodies in the failure of the feasibility of autograft (Dimitriou et al., 2005; Friedlaender et al., 2001; Vaccaro et al., 2002, 2008), and has been approved for the use by FDA. Recently, it has also shown efficacy in various preclinical models of acute and chronic kidney diseases (Zeisberg and Kalluri, 2008). For clinical applications, recombinant human BMP-7 (rhBMP-7) is produced in Chinese hamster ovary (CHO) cells. The rhBMP-7 is synthesized intracellularly as a large precursor. As shown in Fig. 1, it undergoes proteolytic cleavage within the secretory pathway by a subtilisin-like proprotein convertase to secrete the functionally active mature dimer which is the form of rhBMP-7 used for clinical applications. However, the precursor forms of rhBMP-7 are also secreted along with the mature form into the medium, resulting
∗ Corresponding author. Tel.: +82 42 350 2618; fax: +82 42 350 2610. E-mail address: [email protected] (G.M. Lee). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.01.012
in decreased product yield (Israel et al., 1992; Jones et al., 1994). The incomplete proteolytic cleavage of rhBMP-7 is likely due to the insufficient amount of the proteolytic cleaving enzyme within the secretory pathway. A strategy employing the paired basic amino acid cleaving enzyme (PACE) and its derivatives has demonstrated potential to ensure complete proteolytic cleavage of therapeutic proteins. The PACE, a ubiquitously expressed mammalian proprotein convertase, plays an important role in the proteolytic processing of precursor molecules transiting through the constitutive secretory pathway and endocytic pathway (Nakayama, 1997; Roebroek et al., 1994). Expression of full length PACE or a more efficient and secreted form of PACE (PACEsol) in CHO cells improved the processing of recombinant proteins such as human von Willebrand (rhvWF) (Rehemtulla and Kaufman, 1992), factor IX (FIX), and rhBMP-2 (Roe et al., 2004; Wasley et al., 1993). Likewise, PACEsol overexpression in CHO cells may also enhance the processing of rhBMP-7. In the present study, PACEsol was overexpressed in CHO cells producing rhBMP-7 (CHO-BMP-7) in order to increase the product yield and facilitate the purification steps. The proteolytic cleavage site in rhBMP-7 is in accord with the PACE cleavage site (Roebroek et al., 1994). The human PACE cDNA was isolated from HEK293E cells using the following primers: Fp PACE: 5 -ATG GAG CTG AGG CCC TGG
M. Sathyamurthy et al. / Journal of Biotechnology 164 (2012) 336–339
337
Fig. 1. Schematic representation of BMP-7 synthesis and processing (Jones et al., 1994).
TTG-3 , Rp PACE: 5 -TCA GAG GGC GCT CTG GTC TTT-3 . PACEsol was subcloned from the PACE cDNA using the primers Fp PACE and Rp PACEsol: 5 -TCA CTC AGG CAG GTG TGA GG-3 . The PACEsol was then cloned into pcDNA 3.1 (+zeo) (Clontech, Palo Alto, CA), yielding the PACEsol expression vector. The PACEsol and null expression vector were stably transfected to CHO-BMP-7, followed by selection in zeocin, yielding CHO-BMP-7 cells overexpressing PACEsol and null cells, respectively. In order to screen the clones overexpressing PACEsol, CHO-BMP7 cells overexpressing PACEsol were subjected to limiting dilution in 96-well tissue culture plates. Sixty-five stably transfected clones were randomly selected and cultivated in 6-well plates, and the expression levels of human PACEsol in their culture supernatants collected on day 4 were analyzed by Western blot (see supplementary Fig. 1). Among them, forty-one clones showed PACEsol overexpression at varying levels. The null cells used as a negative control showed undetectable levels of PACEsol expression in the culture supernatant. Twenty-one clones expressing high levels of PACEsol were selected and cultivated in 6-well plates (data not shown). Finally, four clones showing high specific growth rates () and specific BMP-7 productivity (qBMP-7 ) (clone #05, #09, #57, and #64) were selected for further analysis. These four clones and null cells were then adapted to grow in a serum-free suspension culture. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2013.01.012. To evaluate the effect of PACEsol overexpression on cell growth, viability and BMP-7 production, clones #05, #09, #57, and #64, and null cells were cultivated in 125 mL flasks containing 50 mL of serum-free medium under agitation at 110 rpm in a humidified 5% CO2 shaking incubator (Adolf Kühner AG, Birsfelden, Switzerland) at 37 ◦ C. Fig. 2 shows the profiles of cell growth, cell viability and BMP7 production. Compared with PACEsol-overexpressing clones, the null cells reached a slightly higher maximum viable cell count (VCC) and then died slightly more rapidly (Fig. 2A and B). The maximum VCC of null cells was (11.1 ± 0.3) × 106 cells/mL (average ± standard
Fig. 2. Profiles of cell growth, cell viability, and BMP-7 production. Clone #05 (closed circle), #09 (open circle), #57 (inverted triangle), #64 (open triangle), and null cells (closed square). (A) Viable cell concentration, (B) cell viability, (C) BMP-7 concentration. Exponentially growing cells were inoculated at an initial cell concentration of 2 × 105 cells/mL in a 125 mL Erlenmeyer flask containing 50 mL of PowerCHO-2 CD (Lonza, Belgium) supplemented with 8 mM glutamine, 70 mg/L dextran sulfate, 2 M methotrexate, and 300 g/mL zeocin. Cell concentration was estimated using a hemocytometer. Viable cells were distinguished from dead cells using the trypan blue dye exclusion method. Error bars represent the standard deviation determined in duplicate experiments.
deviation, n = 2), whereas that of PACEsol-overexpressing clones was in a range of 7.0–9.6 × 106 cells/mL. However, the effect of PACEsol overexpression on was statistically insignificant (n = 2, p > 0.05). The of null cells was 0.77 day−1 , while of PACEsol-overexpressing clones was in a range of 0.54–0.73 day−1 . Accordingly, it appears that overexpression of PACEsol in CHOBMP-7 cells did not affect their cell growth significantly. The BMP-7 concentration in the culture medium was determined using a DuoSet ELISA kit (R&D Systems, Minneapolis,
338
M. Sathyamurthy et al. / Journal of Biotechnology 164 (2012) 336–339
Table 1 The , maximum VCC, qBMP-7 , and maximum BMP-7 concentration of null cells and PACEsol overexpressing CHO BMP-7 clones.a,c Cell lines
(day−1 )
Null #05 #09 #57 #64
0.77 0.73 0.54 0.67 0.70
a b c
± ± ± ± ±
0.00 0.03 0.08 0.05 0.01
Maximum VCC (×106 cells/mL) 11.1 9.6 7.1 7.8 8.2
± ± ± ± ±
0.3 0.1 0.2 0.7 0.7
qBMP-7 (g/106 cells/day)b 0.50 0.58 0.55 0.65 0.64
± ± ± ± ±
0.01 0.02 0.10 0.03 0.02
Maximum BMP-7 titer (g/mL) 8.01 7.75 6.70 7.75 6.48
± ± ± ± ±
0.12 0.86 0.44 0.58 0.52
Values are means ± SD of duplicate experiments. The qBMP-7 was calculated based on the data collected from day 2 to day 5. Data were compared using Student’s two-tailed t-test.
MN). The antibody used in the ELISA kit recognizes all forms of BMP-7 including the processed, unprocessed, and partially processed forms. Thus, the BMP-7 concentration shown in Fig. 2C is a measure of all BMP-7 forms in the culture medium. Regardless of the cell lines used, the BMP-7 concentration in the culture medium reached a maximum on day 6–8, and then decreased until the end of culture, in which the trend is similar to the cell viability (Fig. 2C). There was not a significant statistical difference in the maximum concentration of the null and clones #05 and #57 (n = 2, p > 0.05). However, the qBMP-7 of three clones (#05, #57, and #64) was significantly increased, compared with the null cells (n = 2, p < 0.05). Accordingly, it appears that overexpression of PACEsol in CHO-BMP-7 cells did affect BMP-7 production.
The , maximum VCC, qBMP-7 , and maximum BMP-7 concentration of null cells and PACEsol-overexpressing clones are summarized in Table 1. Among various forms of BMP-7, only the mature form is desired. In order to determine the effect of PACEsol on BMP-7 processing, a Western blot analysis was performed with the culture supernatant from the cultures shown in Fig. 2. Fig. 3A shows Western blots of culture supernatants of null cells and a representative clone #64. The null cells showed the presence of unprocessed and partially or incompletely processed BMP-7 precursor forms. In contrast, clone #64 showed only the mature BMP-7 form, indicating that PACEsol was effective in processing the precursor forms of BMP-7. Other clones also produced only the mature BMP-7 (data not shown).
Fig. 3. Western blot analysis of culture supernatants of null cells and clone #64. (A) An equal volume of the culture medium was separated on 4–12% Bis–Tris NuPAGE gel under non-reducing conditions. (B) Comparison of the molecular weight of mature BMP-7 with (+) and without (−) deglycosylation mix treatment. Membrane was probed with anti-human BMP-7 (Clone 164311; R&D systems, Minneapolis, MN), followed by HRP-conjugated anti-mouse IgG (Koma Biotech, Korea) as secondary antibodies. Bands were visualized using the ECLTM Western blotting detection reagents (GE healthcare, Amersham, Buckinghamshire, UK).
M. Sathyamurthy et al. / Journal of Biotechnology 164 (2012) 336–339
For comparison of the amounts of BMP-7 forms on day 8 between the null cells and clone #64, the band intensity in the Western blots was analyzed using TINA software 2.0 (Raytest). The culture supernatant of clone #64 was homogenous, containing almost 100% of the mature BMP-7 form. On the other hand, that of the null cells consisted of mature BMP-7 (30.9%), ∼45 kDa incompletely processed (33%), ∼63 kDa incompletely processed (28.4%), and ∼94.6 kDa unprocessed-BMP-7 (7.7%). In addition, the decrease in the amounts of mature BMP-7 and the reduction in the molecular weight of the mature BMP7 were evident toward the end of the batch culture (Fig. 3A). Thus, a deglycosylation assay was performed to assess the effect of PACEsol engineering on the protein glycosylation. Fig. 3B shows the Western blots of the culture supernatants of null cells and clone #64 following the deglycosylation assay. In both null and PACEsol clones, a low molecular weight band (∼4 kDa less than the apparent molecular weight, corresponding to the predicted two N-linked glycosylation) was seen on all the days tested, implying PACEsol engineering does not impact the glycosylation of BMP7.However, as the culture progresses, there tends to be a slight shift in the molecular weight of mature BMP-7 toward its deglycosylated form, thus deglycosylation could possibly be one of the cause for the reduced molecular weight of BMP-7 at the end of the batch. Thus, a combination of factors like the degradation by proteases from dead cells and/or the instability under the culture condition and/or deglycosylation of BMP-7 could be responsible for reduced molecular weight and decreased amounts of mature BMP-7 on day 10.Since PACE is ubiquitous present in all mammalian cells, the overexpression of PACEsol is not expected to impact negatively on the activity of mature BMP-7 in CHO cells. The activity of purified BMP-7 was examined, by measuring the alkaline phosphatase activity using MC3T3-E1 cells. BMP-7 from the PACEsol engineered clone was found to be active and comparable to the commercially available BMP-7 (data not shown). It is not still clear whether PACEsol acts only in the culture medium after secretion or it is activated within the secretory compartment of the cell (Bass et al., 2000; Salvas et al., 2005). Nevertheless, PACEsol overexpression in CHO BMP-7 cells was found to eliminate the precursor forms of BMP-7 in the culture medium, yielding significantly improved production of the mature BMP7. This strategy of improving the yield of BMP-7 eliminates the need for removal of the precursors during purification. It could be more beneficial compared to the other strategies that use BMP2/7 chimeric precursor to produce BMP-7 homodimer or the BMP-2/7 synthetic hetero-dimer, with Noggin antagonism (Zhu et al., 2006), which are yet to be proven for the commercial production scale. In conclusion, PACEsol overexpression in CHO cells producing rhBMP-7 was found to be an efficient means of removing the propeptide from the precursor form of BMP-7, increasing the production yield of the mature rhBMP-7, and facilitating its purification. Acknowledgements This research was supported in part by a grant from the Fundamental R&D Program for Technology of World Premier Materials
339
funded by the Ministry of Knowledge Economy, Republic of Korea and the World Class University program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST, R31-2008-000-10071-0). References Bass, J., Turck, C., Rouard, M., Steiner, D.F., 2000. Furin-mediated processing in the early secretory pathway: sequential cleavage and degradation of misfolded insulin receptors. Proceedings of the National Academy of Sciences of the United States of America 97, 11905–11909. Chen, P., Vukicevic, S., Sampath, T.K., Luyten, F.P., 1995. Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures. Journal of Cell Science 108, 105–114. Dimitriou, R., Dahabreh, Z., Katsoulis, E., Matthews, S.J., Branfoot, T., Giannoudis, P.V., 2005. Application of recombinant BMP-7 on persistent upper and lower limb non-unions. Injury 36, S51–S59. Dudley, A.T., Lyons, K.M., Robertson, E.J., 1995. A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes and Development 9, 2795–2807. Friedlaender, G.E., Perry, C.R., Cole, J.D., Cook, S.D., Cierny, G., Muschler, G.F., Zych, G.A., Calhoun, J.H., LaForte, A.J., Yin, S., 2001. Osteogenic protein1 (bone morphogenetic protein-7) in the treatment of tibial nonunions: a prospective, randomized clinical trial comparing rhOP-1 with fresh bone autograft. Journal of Bone and Joint Surgery. American Volume 83-A, S151–S158. Gould, S.E., Day, M., Jones, S.S., Dorai, H., 2002. BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells. Kidney International 61, 51–60. Israel, D.I., Nove, J., Kerns, K.M., Moutsatsos, I.K., Kaufman, R.J., 1992. Expression and characterization of bone morphogenetic protein-2 in chinese hamster ovary cells. Growth Factors 7, 139–150. Jones, W.K., Richmond, E.A., White, K., Sasak, H., Kusmik, W., Smart, J., Oppermann, H., Rueger, D.C., Tucker, F., 1994. Osteogenic protein-1 (OP-1) expression and processing in chinese-hamster ovary cells – isolation of a soluble complex containing the mature and pro-domains of OP-1. Growth Factors 11, 215–225. Nakayama, K., 1997. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochemical Journal 327, 625–635. Rehemtulla, A., Kaufman, R.J., 1992. Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes. Blood 79, 2349–2355. Roebroek, A.J.M., Creemers, J.W.M., Ayoubi, T.A.Y., Van de ven, W.J.M., 1994. Furin-mediated proprotein processing activity: involvement of negatively charged amino-acid-residues in the substrate-binding region. Biochimie 76, 210–216. Roe, S., Francullo, L.E., Paradis, T.M., Porter, T.J., Leonard, M.W., Charlebois, T.S., 2004. Effects of posttranslational processing on rhBMP-2 cellular productivity and product quality. BioProcess International 2, 32–43. Salvas, A., Benjannet, S., Reudelhuber, T.L., Chretien, M., Seidah, N.G., 2005. Evidence for proprotein convertase activity in the endoplasmic reticulum/early Golgi. FEBS Letters 579, 5621–5625. Vaccaro, A.R., Anderson, D.G., Toth, C.A., 2002. Recombinant human osteogenic protein-1 (bone morphogenetic protein-7) as an osteoinductive agent in spinal fusion. Spine 27, S59–S65. Vaccaro, A.R., Whang, P.G., Patel, T., Phillips, F.M., Anderson, D.G., Albert, T.J., Hilibrand, A.S., Brower, R.S., Kurd, M.F., Appannagari, A., Patel, M., Fischgrund, J.S., 2008. The safety and efficacy of OP-1 (rhBMP-7) as a replacement for iliac crest autograft for posterolateral lumbar arthrodesis: minimum 4-year follow-up of a pilot study. Spine Journal 8, 457–465. Wasley, L.C., Rehemtulla, A., Bristol, J.A., Kaufman, R.J., 1993. Pace/furin process the vitamin-K-dependent pro-factor-IX precursor can within the secretory pathway. Journal of Biological Chemistry 268, 8458–8465. Zeisberg, M., Kalluri, R., 2008. Reversal of experimental renal fibrosis by BMP-7 provides insights into novel therapeutic strategies for chronic kidney disease. Pediatric Nephrology 23, 1395–1398. Zhu, W., Kim, J., Cheng, C., Rawlins, B.A., Boachie-Adjei, O., Crystal, R.G., Hidaka, C., 2006. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 39, 61–71.
Contents lists available at SciVerse ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Overexpression of PACEsol improves BMP-7 processing in recombinant CHO cells Madhavi Sathyamurthy a , Jae Seong Lee a , Jin Hyoung Park a , Yeon Jung Kim a , Ji Yeon Jeong b , Ju Woong Jang b , Gyun Min Lee a,∗ a b
Department of Biological Sciences and Graduate School of Nanoscience & Technology (WCU), KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea Institute of Biomaterial and Medical Engineering, Korea Bone Bank, 402 Gasan-dong, Geumcheon-gu, Seoul 153-782, Republic of Korea
a r t i c l e
i n f o
Article history: Received 12 September 2012 Received in revised form 28 December 2012 Accepted 16 January 2013 Available online 31 January 2013 Keywords: BMP-7 BMP-7 precursor CHO cells PACEsol
a b s t r a c t Bone morphogenetic protein-7 (BMP-7) is a member of the TGF- superfamily and plays a critical role in cartilage, bone, and kidney development. BMP-7 is synthesized as a large precursor and undergoes proteolytic cleavage by subtilisin-like proprotein convertase to secrete the functionally active mature dimer. When CHO cells producing recombinant human BMP-7 (rhBMP) (CHO-BMP-7) were cultivated in a serum-free suspension culture, a significant amount of unwanted precursor forms of rhBMP-7 (ca. 69% of total rhBMP-7), along with the mature form of rhBMP-7, was secreted into the culture medium, likely due to the insufficient amount of the proteolytic cleaving enzyme within the secretory pathway. In order to solve this problem, a soluble form of the paired basic amino acid cleaving enzyme (PACEsol), responsible for the majority of the processing events occurring in the constitutive secretory pathway in mammalian cells, was overexpressed in CHO-BMP-7 cells. Overexpression of PACEsol was effective in processing the precursor forms of BMP-7, while it did not significantly affect cell growth. As a result, the culture supernatants of CHO-BMP-7 cells overexpressing PACEsol contained almost 100% of the mature BMP-7 form. Taken together, the results show that PACEsol overexpression in CHO-BMP-7 cells is an efficient means of increasing the production of mature BMP-7 and facilitating the downstream purification steps by eliminating the need to remove the precursor forms. © 2013 Elsevier B.V. All rights reserved.
Bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor- (TGF-) superfamily, is a 35 kDa glycoprotein primarily known for its osteoinductive properties (Chen et al., 1995) and kidney development (Dudley et al., 1995; Gould et al., 2002). Recombinant human BMP-7 (rhBMP-7) has been developed as a potential therapeutic agent for the treatment of tibial nonunion and fusion of vertebral bodies in the failure of the feasibility of autograft (Dimitriou et al., 2005; Friedlaender et al., 2001; Vaccaro et al., 2002, 2008), and has been approved for the use by FDA. Recently, it has also shown efficacy in various preclinical models of acute and chronic kidney diseases (Zeisberg and Kalluri, 2008). For clinical applications, recombinant human BMP-7 (rhBMP-7) is produced in Chinese hamster ovary (CHO) cells. The rhBMP-7 is synthesized intracellularly as a large precursor. As shown in Fig. 1, it undergoes proteolytic cleavage within the secretory pathway by a subtilisin-like proprotein convertase to secrete the functionally active mature dimer which is the form of rhBMP-7 used for clinical applications. However, the precursor forms of rhBMP-7 are also secreted along with the mature form into the medium, resulting
∗ Corresponding author. Tel.: +82 42 350 2618; fax: +82 42 350 2610. E-mail address: [email protected] (G.M. Lee). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.01.012
in decreased product yield (Israel et al., 1992; Jones et al., 1994). The incomplete proteolytic cleavage of rhBMP-7 is likely due to the insufficient amount of the proteolytic cleaving enzyme within the secretory pathway. A strategy employing the paired basic amino acid cleaving enzyme (PACE) and its derivatives has demonstrated potential to ensure complete proteolytic cleavage of therapeutic proteins. The PACE, a ubiquitously expressed mammalian proprotein convertase, plays an important role in the proteolytic processing of precursor molecules transiting through the constitutive secretory pathway and endocytic pathway (Nakayama, 1997; Roebroek et al., 1994). Expression of full length PACE or a more efficient and secreted form of PACE (PACEsol) in CHO cells improved the processing of recombinant proteins such as human von Willebrand (rhvWF) (Rehemtulla and Kaufman, 1992), factor IX (FIX), and rhBMP-2 (Roe et al., 2004; Wasley et al., 1993). Likewise, PACEsol overexpression in CHO cells may also enhance the processing of rhBMP-7. In the present study, PACEsol was overexpressed in CHO cells producing rhBMP-7 (CHO-BMP-7) in order to increase the product yield and facilitate the purification steps. The proteolytic cleavage site in rhBMP-7 is in accord with the PACE cleavage site (Roebroek et al., 1994). The human PACE cDNA was isolated from HEK293E cells using the following primers: Fp PACE: 5 -ATG GAG CTG AGG CCC TGG
M. Sathyamurthy et al. / Journal of Biotechnology 164 (2012) 336–339
337
Fig. 1. Schematic representation of BMP-7 synthesis and processing (Jones et al., 1994).
TTG-3 , Rp PACE: 5 -TCA GAG GGC GCT CTG GTC TTT-3 . PACEsol was subcloned from the PACE cDNA using the primers Fp PACE and Rp PACEsol: 5 -TCA CTC AGG CAG GTG TGA GG-3 . The PACEsol was then cloned into pcDNA 3.1 (+zeo) (Clontech, Palo Alto, CA), yielding the PACEsol expression vector. The PACEsol and null expression vector were stably transfected to CHO-BMP-7, followed by selection in zeocin, yielding CHO-BMP-7 cells overexpressing PACEsol and null cells, respectively. In order to screen the clones overexpressing PACEsol, CHO-BMP7 cells overexpressing PACEsol were subjected to limiting dilution in 96-well tissue culture plates. Sixty-five stably transfected clones were randomly selected and cultivated in 6-well plates, and the expression levels of human PACEsol in their culture supernatants collected on day 4 were analyzed by Western blot (see supplementary Fig. 1). Among them, forty-one clones showed PACEsol overexpression at varying levels. The null cells used as a negative control showed undetectable levels of PACEsol expression in the culture supernatant. Twenty-one clones expressing high levels of PACEsol were selected and cultivated in 6-well plates (data not shown). Finally, four clones showing high specific growth rates () and specific BMP-7 productivity (qBMP-7 ) (clone #05, #09, #57, and #64) were selected for further analysis. These four clones and null cells were then adapted to grow in a serum-free suspension culture. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2013.01.012. To evaluate the effect of PACEsol overexpression on cell growth, viability and BMP-7 production, clones #05, #09, #57, and #64, and null cells were cultivated in 125 mL flasks containing 50 mL of serum-free medium under agitation at 110 rpm in a humidified 5% CO2 shaking incubator (Adolf Kühner AG, Birsfelden, Switzerland) at 37 ◦ C. Fig. 2 shows the profiles of cell growth, cell viability and BMP7 production. Compared with PACEsol-overexpressing clones, the null cells reached a slightly higher maximum viable cell count (VCC) and then died slightly more rapidly (Fig. 2A and B). The maximum VCC of null cells was (11.1 ± 0.3) × 106 cells/mL (average ± standard
Fig. 2. Profiles of cell growth, cell viability, and BMP-7 production. Clone #05 (closed circle), #09 (open circle), #57 (inverted triangle), #64 (open triangle), and null cells (closed square). (A) Viable cell concentration, (B) cell viability, (C) BMP-7 concentration. Exponentially growing cells were inoculated at an initial cell concentration of 2 × 105 cells/mL in a 125 mL Erlenmeyer flask containing 50 mL of PowerCHO-2 CD (Lonza, Belgium) supplemented with 8 mM glutamine, 70 mg/L dextran sulfate, 2 M methotrexate, and 300 g/mL zeocin. Cell concentration was estimated using a hemocytometer. Viable cells were distinguished from dead cells using the trypan blue dye exclusion method. Error bars represent the standard deviation determined in duplicate experiments.
deviation, n = 2), whereas that of PACEsol-overexpressing clones was in a range of 7.0–9.6 × 106 cells/mL. However, the effect of PACEsol overexpression on was statistically insignificant (n = 2, p > 0.05). The of null cells was 0.77 day−1 , while of PACEsol-overexpressing clones was in a range of 0.54–0.73 day−1 . Accordingly, it appears that overexpression of PACEsol in CHOBMP-7 cells did not affect their cell growth significantly. The BMP-7 concentration in the culture medium was determined using a DuoSet ELISA kit (R&D Systems, Minneapolis,
338
M. Sathyamurthy et al. / Journal of Biotechnology 164 (2012) 336–339
Table 1 The , maximum VCC, qBMP-7 , and maximum BMP-7 concentration of null cells and PACEsol overexpressing CHO BMP-7 clones.a,c Cell lines
(day−1 )
Null #05 #09 #57 #64
0.77 0.73 0.54 0.67 0.70
a b c
± ± ± ± ±
0.00 0.03 0.08 0.05 0.01
Maximum VCC (×106 cells/mL) 11.1 9.6 7.1 7.8 8.2
± ± ± ± ±
0.3 0.1 0.2 0.7 0.7
qBMP-7 (g/106 cells/day)b 0.50 0.58 0.55 0.65 0.64
± ± ± ± ±
0.01 0.02 0.10 0.03 0.02
Maximum BMP-7 titer (g/mL) 8.01 7.75 6.70 7.75 6.48
± ± ± ± ±
0.12 0.86 0.44 0.58 0.52
Values are means ± SD of duplicate experiments. The qBMP-7 was calculated based on the data collected from day 2 to day 5. Data were compared using Student’s two-tailed t-test.
MN). The antibody used in the ELISA kit recognizes all forms of BMP-7 including the processed, unprocessed, and partially processed forms. Thus, the BMP-7 concentration shown in Fig. 2C is a measure of all BMP-7 forms in the culture medium. Regardless of the cell lines used, the BMP-7 concentration in the culture medium reached a maximum on day 6–8, and then decreased until the end of culture, in which the trend is similar to the cell viability (Fig. 2C). There was not a significant statistical difference in the maximum concentration of the null and clones #05 and #57 (n = 2, p > 0.05). However, the qBMP-7 of three clones (#05, #57, and #64) was significantly increased, compared with the null cells (n = 2, p < 0.05). Accordingly, it appears that overexpression of PACEsol in CHO-BMP-7 cells did affect BMP-7 production.
The , maximum VCC, qBMP-7 , and maximum BMP-7 concentration of null cells and PACEsol-overexpressing clones are summarized in Table 1. Among various forms of BMP-7, only the mature form is desired. In order to determine the effect of PACEsol on BMP-7 processing, a Western blot analysis was performed with the culture supernatant from the cultures shown in Fig. 2. Fig. 3A shows Western blots of culture supernatants of null cells and a representative clone #64. The null cells showed the presence of unprocessed and partially or incompletely processed BMP-7 precursor forms. In contrast, clone #64 showed only the mature BMP-7 form, indicating that PACEsol was effective in processing the precursor forms of BMP-7. Other clones also produced only the mature BMP-7 (data not shown).
Fig. 3. Western blot analysis of culture supernatants of null cells and clone #64. (A) An equal volume of the culture medium was separated on 4–12% Bis–Tris NuPAGE gel under non-reducing conditions. (B) Comparison of the molecular weight of mature BMP-7 with (+) and without (−) deglycosylation mix treatment. Membrane was probed with anti-human BMP-7 (Clone 164311; R&D systems, Minneapolis, MN), followed by HRP-conjugated anti-mouse IgG (Koma Biotech, Korea) as secondary antibodies. Bands were visualized using the ECLTM Western blotting detection reagents (GE healthcare, Amersham, Buckinghamshire, UK).
M. Sathyamurthy et al. / Journal of Biotechnology 164 (2012) 336–339
For comparison of the amounts of BMP-7 forms on day 8 between the null cells and clone #64, the band intensity in the Western blots was analyzed using TINA software 2.0 (Raytest). The culture supernatant of clone #64 was homogenous, containing almost 100% of the mature BMP-7 form. On the other hand, that of the null cells consisted of mature BMP-7 (30.9%), ∼45 kDa incompletely processed (33%), ∼63 kDa incompletely processed (28.4%), and ∼94.6 kDa unprocessed-BMP-7 (7.7%). In addition, the decrease in the amounts of mature BMP-7 and the reduction in the molecular weight of the mature BMP7 were evident toward the end of the batch culture (Fig. 3A). Thus, a deglycosylation assay was performed to assess the effect of PACEsol engineering on the protein glycosylation. Fig. 3B shows the Western blots of the culture supernatants of null cells and clone #64 following the deglycosylation assay. In both null and PACEsol clones, a low molecular weight band (∼4 kDa less than the apparent molecular weight, corresponding to the predicted two N-linked glycosylation) was seen on all the days tested, implying PACEsol engineering does not impact the glycosylation of BMP7.However, as the culture progresses, there tends to be a slight shift in the molecular weight of mature BMP-7 toward its deglycosylated form, thus deglycosylation could possibly be one of the cause for the reduced molecular weight of BMP-7 at the end of the batch. Thus, a combination of factors like the degradation by proteases from dead cells and/or the instability under the culture condition and/or deglycosylation of BMP-7 could be responsible for reduced molecular weight and decreased amounts of mature BMP-7 on day 10.Since PACE is ubiquitous present in all mammalian cells, the overexpression of PACEsol is not expected to impact negatively on the activity of mature BMP-7 in CHO cells. The activity of purified BMP-7 was examined, by measuring the alkaline phosphatase activity using MC3T3-E1 cells. BMP-7 from the PACEsol engineered clone was found to be active and comparable to the commercially available BMP-7 (data not shown). It is not still clear whether PACEsol acts only in the culture medium after secretion or it is activated within the secretory compartment of the cell (Bass et al., 2000; Salvas et al., 2005). Nevertheless, PACEsol overexpression in CHO BMP-7 cells was found to eliminate the precursor forms of BMP-7 in the culture medium, yielding significantly improved production of the mature BMP7. This strategy of improving the yield of BMP-7 eliminates the need for removal of the precursors during purification. It could be more beneficial compared to the other strategies that use BMP2/7 chimeric precursor to produce BMP-7 homodimer or the BMP-2/7 synthetic hetero-dimer, with Noggin antagonism (Zhu et al., 2006), which are yet to be proven for the commercial production scale. In conclusion, PACEsol overexpression in CHO cells producing rhBMP-7 was found to be an efficient means of removing the propeptide from the precursor form of BMP-7, increasing the production yield of the mature rhBMP-7, and facilitating its purification. Acknowledgements This research was supported in part by a grant from the Fundamental R&D Program for Technology of World Premier Materials
339
funded by the Ministry of Knowledge Economy, Republic of Korea and the World Class University program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST, R31-2008-000-10071-0). References Bass, J., Turck, C., Rouard, M., Steiner, D.F., 2000. Furin-mediated processing in the early secretory pathway: sequential cleavage and degradation of misfolded insulin receptors. Proceedings of the National Academy of Sciences of the United States of America 97, 11905–11909. Chen, P., Vukicevic, S., Sampath, T.K., Luyten, F.P., 1995. Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures. Journal of Cell Science 108, 105–114. Dimitriou, R., Dahabreh, Z., Katsoulis, E., Matthews, S.J., Branfoot, T., Giannoudis, P.V., 2005. Application of recombinant BMP-7 on persistent upper and lower limb non-unions. Injury 36, S51–S59. Dudley, A.T., Lyons, K.M., Robertson, E.J., 1995. A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes and Development 9, 2795–2807. Friedlaender, G.E., Perry, C.R., Cole, J.D., Cook, S.D., Cierny, G., Muschler, G.F., Zych, G.A., Calhoun, J.H., LaForte, A.J., Yin, S., 2001. Osteogenic protein1 (bone morphogenetic protein-7) in the treatment of tibial nonunions: a prospective, randomized clinical trial comparing rhOP-1 with fresh bone autograft. Journal of Bone and Joint Surgery. American Volume 83-A, S151–S158. Gould, S.E., Day, M., Jones, S.S., Dorai, H., 2002. BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells. Kidney International 61, 51–60. Israel, D.I., Nove, J., Kerns, K.M., Moutsatsos, I.K., Kaufman, R.J., 1992. Expression and characterization of bone morphogenetic protein-2 in chinese hamster ovary cells. Growth Factors 7, 139–150. Jones, W.K., Richmond, E.A., White, K., Sasak, H., Kusmik, W., Smart, J., Oppermann, H., Rueger, D.C., Tucker, F., 1994. Osteogenic protein-1 (OP-1) expression and processing in chinese-hamster ovary cells – isolation of a soluble complex containing the mature and pro-domains of OP-1. Growth Factors 11, 215–225. Nakayama, K., 1997. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochemical Journal 327, 625–635. Rehemtulla, A., Kaufman, R.J., 1992. Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes. Blood 79, 2349–2355. Roebroek, A.J.M., Creemers, J.W.M., Ayoubi, T.A.Y., Van de ven, W.J.M., 1994. Furin-mediated proprotein processing activity: involvement of negatively charged amino-acid-residues in the substrate-binding region. Biochimie 76, 210–216. Roe, S., Francullo, L.E., Paradis, T.M., Porter, T.J., Leonard, M.W., Charlebois, T.S., 2004. Effects of posttranslational processing on rhBMP-2 cellular productivity and product quality. BioProcess International 2, 32–43. Salvas, A., Benjannet, S., Reudelhuber, T.L., Chretien, M., Seidah, N.G., 2005. Evidence for proprotein convertase activity in the endoplasmic reticulum/early Golgi. FEBS Letters 579, 5621–5625. Vaccaro, A.R., Anderson, D.G., Toth, C.A., 2002. Recombinant human osteogenic protein-1 (bone morphogenetic protein-7) as an osteoinductive agent in spinal fusion. Spine 27, S59–S65. Vaccaro, A.R., Whang, P.G., Patel, T., Phillips, F.M., Anderson, D.G., Albert, T.J., Hilibrand, A.S., Brower, R.S., Kurd, M.F., Appannagari, A., Patel, M., Fischgrund, J.S., 2008. The safety and efficacy of OP-1 (rhBMP-7) as a replacement for iliac crest autograft for posterolateral lumbar arthrodesis: minimum 4-year follow-up of a pilot study. Spine Journal 8, 457–465. Wasley, L.C., Rehemtulla, A., Bristol, J.A., Kaufman, R.J., 1993. Pace/furin process the vitamin-K-dependent pro-factor-IX precursor can within the secretory pathway. Journal of Biological Chemistry 268, 8458–8465. Zeisberg, M., Kalluri, R., 2008. Reversal of experimental renal fibrosis by BMP-7 provides insights into novel therapeutic strategies for chronic kidney disease. Pediatric Nephrology 23, 1395–1398. Zhu, W., Kim, J., Cheng, C., Rawlins, B.A., Boachie-Adjei, O., Crystal, R.G., Hidaka, C., 2006. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 39, 61–71.