Aileen J Zhou

Cameron ML Clokie

JM Wozney

Sean AF Peel

Y Cui

English

PubMed

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MEETING ABSTRACT Open Access
A novel peptide to enhance recombinant BMP-2
production in mammalian cell cultures
Aileen J Zhou1*, Cameron ML Clokie1,2, Sean AF Peel1,2
From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies
Vienna, Austria. 15-18 May 2011
Background
Due to their osteoinductive properties, recombinant
human bone morphogenetic proteins (rhBMPs) have
been used successfully for bone regeneration and repla-
cement. However, the yields rhBMPs yields in mamma-
lian expression systems are very low, resulting in their
high cost. BMPs are synthesized as a precursor,
proBMP, which undergoes enzymatic cleavage by pro-
protein convertases (PCs) to form the mature BMP [1].
Furin, an enzyme of the PC family, has shown to cleave
BMP-4 [2] and BMP-2 (Zhou et al., unpublished data).
This study investigated the effect and mechanism of
action of polyarginine furin inhibitor, IND-1, on
rhBMP-2 production in mammalian cell lines overex-
pressing rhBMP-2.
Materials and methods
Two stable cell lines expressing the hBMP2 gene, CHO-
BMP2 and HEK-BMP2, were cultured in the presence
of IND-1 in short-term (24 h, multi-well) and long-term
(two-month, perfusion flasks) cultures. The rhBMP-2
produced was characterized by Western blot and its
activity assessed using the C2C12 cell-based assay. The
amount of proBMP-2 and mature BMP-2 produced was
quantified by ELISA. The mRNA level of BMP-2 and
furin in cells treated with or without IND-1 was com-
pared by real-time RT-PCR. Cellular uptake of IND-1
was estimated by measuring the fluorescence of cell
lysates following incubation with FITC labeled IND-1.
Cellular PC activity post IND-1 incubation was mea-
sured using the Boc-RVRR-AMC substrate. Furin-speci-
fic siRNA was used to knock down the furin expression
in CHO-BMP2 cells and its effect on the rhBMP-2 pro-
duction was determined.
Results
Stably transfected CHO-BMP2 cells secreted 36 kDa
rhBMP-2 dimers that were biologically active. In 24 h
cell cultures, IND-1 treated cells produced significantly
greater amounts of proBMP-2 (≥ 10-fold, P <0.001) and
mature BMP-2 (≥ 3-fold, P <0.001) in their conditioned
medium (Figure 1). In long-term CHO-BMP2 culture,
IND-1 continued to increase the yields of BMP-2 (≥
50%) and proBMP-2 (≥ 2-fold) without affecting cell
growth or viability.
IND-1 treatment had no effect on the mRNA level of
BMP-2 and furin, indicating IND-1 affects rhBMP-2
yield post-transcriptionally. While IND-1 was taken up
by the cells and inhibited PC activity when added
directly to the cell lysates, cells cultured with IND-1
showed no changes in their PC activity at doses 50
times higher than required to affect BMP-2 yields.
Furthermore, knockdown of furin at both the mRNA (≥
80%, P <0.001) and the protein level (≥ 70%, P <0.001),
did not affect rhBMP-2 yields. These results suggest that
furin inhibition is most likely not the mechanism by
which IND-1 enhances rhBMP-2 yields.
Conclusions
The addition of a novel peptide IND-1 to the cell cul-
ture medium significantly enhanced the yields of both
pro- and mature BMP-2 in stably transfected CHO and
HEK cell lines. These increases were sustainable over an
extended time period with regular IND-1 treatments.
However, the enhanced rhBMP-2 yield is unlikely due
to the well-established role of polyarginines as furin
inhibitors.
* Correspondence: aileen.zhou@dentistry.utoronto.ca
1Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University
of Toronto, Toronto, Ontario Canada, M5G 1G6
Full list of author information is available at the end of the article
Zhou et al. BMC Proceedings 2011, 5(Suppl 8):P96
http://www.biomedcentral.com/1753-6561/5/S8/P96
© 2011 Zhou et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Acknowledgements
This work was supported by Oral and Maxillofacial Surgery Foundation of
Canada and Induce Biologics Inc.
Author details
1Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University
of Toronto, Toronto, Ontario Canada, M5G 1G6. 2Induce Biologics Inc,
Toronto, Ontario, Canada, M5R 3N8.
Published: 22 November 2011
References
1. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitter MJ, Kriz RW,
Hewick RM, Wang EA: Novel regulators of bone-formation-Molecular
clones and activities. Science 1988, 242:1528-1534.
2. Cui Y, Jean F, Thomas G, Christian JL: BMP-4 is proteolytically activated by
furin and/or PC6 during vertebrate embryonic development. EMBO J
1998, 17:4735-4743.
doi:10.1186/1753-6561-5-S8-P96
Cite this article as: Zhou et al.: A novel peptide to enhance
recombinant BMP-2 production in mammalian cell cultures. BMC
Proceedings 2011 5(Suppl 8):P96.
Figure 1 After 24 h incubation with or without IND-1, the amount of proBMP-2 and mature BMP-2 proteins in (A) CHO and (B) HEK conditioned
media were measured by proBMP-2 and BMP-2 ELISA. Data are presented as mean ± SEM (n = 3 experiments) (**P <0.01, ***P <0.001).
Zhou et al. BMC Proceedings 2011, 5(Suppl 8):P96
http://www.biomedcentral.com/1753-6561/5/S8/P96
Page 2 of 2


A novel peptide to enhance recombinant BMP-2 production in mammalian cell cultures

Zhou Aileen J

Clokie Cameron ML

Peel Sean AF

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MEETING ABSTRACT Open AccessA novel peptide to enhance recombinant BMP-2production in mammalian cell culturesAileen J Zhou1*, Cameron ML Clokie1,2, Sean AF Peel1,2From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based TechnologiesVienna, Austria. 15-18 May 2011BackgroundDue to their osteoinductive properties, recombinanthuman bone morphogenetic proteins (rhBMPs) havebeen used successfully for bone regeneration and repla-cement. However, the yields rhBMPs yields in mamma-lian expression systems are very low, resulting in theirhigh cost. BMPs are synthesized as a precursor,proBMP, which undergoes enzymatic cleavage by pro-protein convertases (PCs) to form the mature BMP [1].Furin, an enzyme of the PC family, has shown to cleaveBMP-4 [2] and BMP-2 (Zhou et al., unpublished data).This study investigated the effect and mechanism ofaction of polyarginine furin inhibitor, IND-1, onrhBMP-2 production in mammalian cell lines overex-pressing rhBMP-2.Materials and methodsTwo stable cell lines expressing the hBMP2 gene, CHO-BMP2 and HEK-BMP2, were cultured in the presenceof IND-1 in short-term (24 h, multi-well) and long-term(two-month, perfusion flasks) cultures. The rhBMP-2produced was characterized by Western blot and itsactivity assessed using the C2C12 cell-based assay. Theamount of proBMP-2 and mature BMP-2 produced wasquantified by ELISA. The mRNA level of BMP-2 andfurin in cells treated with or without IND-1 was com-pared by real-time RT-PCR. Cellular uptake of IND-1was estimated by measuring the fluorescence of celllysates following incubation with FITC labeled IND-1.Cellular PC activity post IND-1 incubation was mea-sured using the Boc-RVRR-AMC substrate. Furin-speci-fic siRNA was used to knock down the furin expressionin CHO-BMP2 cells and its effect on the rhBMP-2 pro-duction was determined.ResultsStably transfected CHO-BMP2 cells secreted 36 kDarhBMP-2 dimers that were biologically active. In 24 hcell cultures, IND-1 treated cells produced significantlygreater amounts of proBMP-2 (≥ 10-fold, P <0.001) andmature BMP-2 (≥ 3-fold, P <0.001) in their conditionedmedium (Figure 1). In long-term CHO-BMP2 culture,IND-1 continued to increase the yields of BMP-2 (≥50%) and proBMP-2 (≥ 2-fold) without affecting cellgrowth or viability.IND-1 treatment had no effect on the mRNA level ofBMP-2 and furin, indicating IND-1 affects rhBMP-2yield post-transcriptionally. While IND-1 was taken upby the cells and inhibited PC activity when addeddirectly to the cell lysates, cells cultured with IND-1showed no changes in their PC activity at doses 50times higher than required to affect BMP-2 yields.Furthermore, knockdown of furin at both the mRNA (≥80%, P <0.001) and the protein level (≥ 70%, P <0.001),did not affect rhBMP-2 yields. These results suggest thatfurin inhibition is most likely not the mechanism bywhich IND-1 enhances rhBMP-2 yields.ConclusionsThe addition of a novel peptide IND-1 to the cell cul-ture medium significantly enhanced the yields of bothpro- and mature BMP-2 in stably transfected CHO andHEK cell lines. These increases were sustainable over anextended time period with regular IND-1 treatments.However, the enhanced rhBMP-2 yield is unlikely dueto the well-established role of polyarginines as furininhibitors.* Correspondence: aileen.zhou@dentistry.utoronto.ca1Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Universityof Toronto, Toronto, Ontario Canada, M5G 1G6Full list of author information is available at the end of the articleZhou et al. BMC Proceedings 2011, 5(Suppl 8):P96http://www.biomedcentral.com/1753-6561/5/S8/P96© 2011 Zhou et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.AcknowledgementsThis work was supported by Oral and Maxillofacial Surgery Foundation ofCanada and Induce Biologics Inc.Author details1Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Universityof Toronto, Toronto, Ontario Canada, M5G 1G6. 2Induce Biologics Inc,Toronto, Ontario, Canada, M5R 3N8.Published: 22 November 2011References1. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitter MJ, Kriz RW,Hewick RM, Wang EA: Novel regulators of bone-formation-Molecularclones and activities. Science 1988, 242:1528-1534.2. Cui Y, Jean F, Thomas G, Christian JL: BMP-4 is proteolytically activated byfurin and/or PC6 during vertebrate embryonic development. EMBO J1998, 17:4735-4743.doi:10.1186/1753-6561-5-S8-P96Cite this article as: Zhou et al.: A novel peptide to enhancerecombinant BMP-2 production in mammalian cell cultures. BMCProceedings 2011 5(Suppl 8):P96.Figure 1 After 24 h incubation with or without IND-1, the amount of proBMP-2 and mature BMP-2 proteins in (A) CHO and (B) HEK conditionedmedia were measured by proBMP-2 and BMP-2 ELISA. Data are presented as mean ± SEM (n = 3 experiments) (**P <0.01, ***P <0.001).Zhou et al. BMC Proceedings 2011, 5(Suppl 8):P96http://www.biomedcentral.com/1753-6561/5/S8/P96Page 2 of 2

Zhou, Aileen J

Clokie, Cameron M

Peel, Sean A

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EA: Novel regulators of bone-formation-Molecular clones and activities. Science

JL: BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development.

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A novel peptide to enhance recombinant BMP-2 production in mammalian cell cultures

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