Biotechnol Lett (2012) 34:221230 DOI 10.

1007/s10529-011-0781-7

ORIGINAL RESEARCH PAPER

Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells
Aileen J. Zhou Cameron M. L. Clokie Sean A. F. Peel

Received: 19 September 2011 / Accepted: 12 October 2011 / Published online: 22 October 2011 Springer Science+Business Media B.V. 2011

Abstract To improve recombinant human bone morphogenetic protein-2 (rhBMP-2) yield, cell lines stably expressing hBMP2 were cultured in the presence of polyarginine peptide IND-1 and showed up to 6-fold increase in the yield of mature BMP-2. Repeated addition of IND-1 to cell cultures consistently improved BMP-2 yields over 53 days without affecting cell growth and viability. Investigation of its mechanism of action showed that IND-1 inhibited pro-protein convertase (PC) activity when incubated with cell lysates. However, when intact cells were cultured with IND-1, no change in cellular PC activity was observed.

Furthermore, knockdown of furin (a prototypical member of the PCs) in cells did not affect their BMP-2 yields, suggesting furin/PC inhibition is unlikely the mechanism by which IND-1 enhances BMP-2 yields. IND-1 as a medium additive thus enhances BMP-2 production in mammalian cell expression systems. Keywords Bone morphogenetic protein Polyarginine Proprotein convertase Recombinant protein

Introduction
A patent application has been led describing methods that enhance the production of recombinant proteins, which includes the methods described in this paper (Methods for enhancing recombinant protein production (PCT/CA2008/ 000998)). This pending patent application has been assigned to Induce Biologics Inc. SAFP and CMLC are shareholders in Induce Biologics Inc. SAFP is an employee of Induce Biologics Inc.

Electronic supplementary material The online version of this article (doi:10.1007/s10529-011-0781-7) contains supplementary material, which is available to authorized users.
A. J. Zhou C. M. L. Clokie S. A. F. Peel (&) Oral & Maxillofacial Surgery, Faculty of Dentistry, University of Toronto, 124 Edward Street, Room 412, Toronto, Toronto, ON M5G 1G6, Canada e-mail: sean.peel@dentistry.utoronto.ca S. A. F. Peel Induce Biologics Inc., Toronto, ON, Canada

Bone morphogenetic proteins (BMPs) are a sub-group of growth factors within the TGF-b superfamily (Wozney et al. 1988). Among its members, BMP-2 is best known for its strong ability to induce bone formation (Urist 1965). Native BMP-2 is initially synthesized as a large precursor, proBMP-2, which undergoes dimerization and enzymatic cleavage to generate the mature BMP-2 dimers (Wozney et al. 1988). The enzymes responsible for cleaving BMP precursors are believed to be furin and furin-like proprotein convertases (PCs) (Cui et al. 1998; Heng et al. 2010; Nelsen and Christian 2009). Due to its osteoinductive ability, BMP-2 has been frequently used in tissue engineering products for bone regeneration. Initially, BMPs were isolated directly from bones but the yields were low and the process

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was labourious (Bessho et al. 1989; Hu et al. 2004). Recombinant human BMPs (rhBMPs) were produced from Escherichia coli (Hillger et al. 2005; Yano et al. 2009) and mammalian cell culture systems (Israel et al. 1992; Wang et al. 1990; Yamaguchi et al. 1991). Despite successfully producing properly folded, glycosylated and biologically active rhBMP-2, the yield of mammalian cell expression system is low, that remains a major challenge and hinders the use of rhBMP-2 in therapeutic applications. A preliminary study from our laboratory where we cultivated cells in the presence of polyarginine peptides has generated interesting results (Zhou et al. 2009). The aim of the present work investigated the potential of using a polyarginine peptide, IND-1, as a simple cell culture media additive to improve rhBMP2 yield. Here we demonstrated that IND-1 effectively increased the rhBMP-2 yields in CHO and HEK cell lines without affecting cell growth or viability. In addition, we investigated IND-10 s mechanism of action on rhBMP-2 yield.

and 80 lM IND-1 (Induce Biologics Inc., Toronto, Canada), a poly-D-arginine peptide. After 24 h, the conditioned media were collected and centrifuged at 5009g for 5 min to remove any cells. In the long-term study, two CELLine CL350 perfusion cell culture asks (Mandel Scientic, Guelph, Canada) with separate cell and medium compartments were used. CHO-BMP2 cells cultured in CELLine asks with or without IND-1 over 53 days. Conditioned medium from the cell compartment was harvested every 34 days and the media reservoir was replenished once a week. Cells were sub-cultured every 34 days by seeding 50% of recovered cells into cell compartment after ltered by a 100 lm cell sieve (Thermo Fisher Scientic) to remove cell clumps. Cell density and viability analysis Cell density and viability was determined using Vi-CELL XR cell analyzer (Beckman Coulter, Mississauga, Canada). Cell density was determined by cell count of 100 video images captured per sample using the Vi-CELL 1.00 software (Beckman) and cell viability was determined using the Trypan Blue exclusion method. Detection of rhBMP-2 by western blot CHO-BMP2 cells (106 cells/ml) were cultured in T75 asks with serum-free chemically dened OptiCHO Medium (Invitrogen), since serum proteins in FBS interfered with the concentrating of the samples. After 72 h, conditioned medium was collected and concentrated 5- to 7-fold using Amicon Ultra lters (3000 MWCO, Millipore). The concentrated samples were resolved by 412% (v/v) SDS-PAGE gel (Invitrogen) under non-reducing condition and then transferred to a nitrocellulose membrane at 170 mA and 30 V for 2 h. The membranes were blocked with 5% (w/v) BSA in PBS-T buffer (PBS, 0.05% Tween 20) and probed rst with monoclonal mouse anti-BMP-2 antibody (2 lg/ml, BAM3552, R&D Systems) and then with a horseradish peroxidase-linked anti-mouse antibody (Santa Cruz Biotechnology Inc., Santa Cruz, USA), diluted 1:2000. Signal was visualized by incubating with SuperSignal chemiluminescent substrate (Pierce, Thermo Fisher Scientic). Image detection was performed with ChemiDox XRS and Quantity One software (BioRad).

Materials and methods Recombinant expression of hBMP-2 The Flp-In Chinese hamster ovarian (CHO) and human embryonic kidney-293 (HEK) cell lines (Invitrogen) were used to produce the recombinant human BMP-2 (rhBMP-2). Previously, the Flp-In system has been used to successfully express monoclonal antibodies in CHO cells (Huang et al. 2007; Wiberg et al. 2006) and hemagglutinin proteins in HEK cells (Lu et al. 2011). The Flp-In cell lines were stably transfected with pcDNA5/FRT (where FRT is the Flp (ippase) recombination target) containing the cDNA encoding the full-length human proBMP-2 (amino acids 1396), generating CHO-BMP2 and HEK-BMP2 cell lines. Cell culture CHO-BMP2 and HEK-BMP2 cell lines were cultured in a-minimum essential medium (a-MEM) plus 10% fetal bovine serum (FBS) at 37C with 5% CO2. Both cell lines were grown in 6-well plates (2.5 9 105 cells/ well) until 90% conuent. The media was then replaced with test media containing 0, 10, 20, 40,

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Sandwich enzyme-linked immunosorbent assay (ELISA) Mature rhBMP-2 in the cell culture medium was measured using Quantikine BMP-2 immunoassay (R&D Systems), following the manufacturers instructions. This ELISA demonstrated no cross-reactivity (\0.2%) for human proBMP-2 (Axxora, San Diego, USA) and was thus considered specic for mature rhBMP-2. For measuring the proBMP-2 in the conditioned, a microplate was coated with monoclonal antiproBMP-2 antibodies (1 lg/ml, MAB2260, R&D systems). This antibody detects the pro-domain of the human proBMP-2 and does not recognize the mature BMP-2 (according to the suppliers datasheet), and thus is considered specic for capturing proBMP2 proteins. The captured proBMP-2 was detected by anti-BMP-2 detection antibody from the Quantikine BMP-2 immunoassay kit. A recombinant human proBMP-2 (Axxora) was used to construct a standard curve. The amount of furin in the cell lysate was determined using a furin DuoSet ELISA Development System (R&D Systems) following manufacturers instructions. BMP-2 bioactivity assay C2C12 cells (ATCC) were seeded at 0.5 9 105 cells per well in a-MEM plus 10% (v/v) FBS in 24-well plates and allowed to attached overnight. Cells were incubated for 48 h with CHO-BMP2 conditioned media and media containing a CHO-produced rhBMP-2 from the Infuse kit (Medtronic, Memphis, USA), which served as control. After incubation, cells were lysed, homogenized and the alkaline phosphatase (ALP) enzymatic activity was determined using paranitrophenyl phosphate (pNPP) as previously described (Peel et al. 2003). The ALP activity was normalized to the amount of cellular protein determined by Coomassie Plus assay (Fischer Scientic). Furin knockdown by siRNA Three pairs of small interfering RNA (siRNA) targeting the furin gene (F1, F2 and F3) and two non-specic siRNAs (with or without 50 FAM label, used as negative controls to determine non-specic genesilencing effects) were synthesized by Shanghai GenePharma Company (Shanghai, China) (Table 1).

HEK-BMP2 cells were plated at 1.25 9 105 cells per well in 6-well plates. After 48 h, the cells were transfected with 20 lM furin specic or non-specic siRNAs using Lipofectamin 2000 (Invitrogen), according to manufacturers instructions. Cell lysates and the conditioned media were collected post transfection. The extent of furin knockdown was assessed by quantifying furin gene expression by real time RT-PCR and protein expression by furin ELISA (described previously). RNA isolation and analysis by quantitative RT-PCR Total RNA from the cells was extracted and DNase treated using RNeasy spin columns (Qiagen). The quantity and purity of extract RNA were determined by spectrophotometry at 260 and 280 nm. Quantitative RT-PCR was performed using QuantiTect SYBR Green One Step RT-PCR reaction mixture (Qiagen) according to manufacturers instructions and detected by iQ 5 multicolor real-time PCR detection system (Bio-Rad) using the following program: 30 min reverse transcription at 50C, 15 min activation for 95C, followed by 40 cycles of 15 s at 94C denaturation, 30 s at 60C anealing and 30 s at 72C for extension. After completion, melting curve analysis was recorded from 55 to 95C at a ramp rate of 0.05 C/s. All experiments included controls (samples without RNA or primers) and the samples were prepared in triplicate, ran in 20 ll qPCR reactions. All target gene transcripts were normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH). Real-time PCR primers were designed using MacVector (MacVector Inc., Cary, USA) (Table 2). All the primers span at least one intron to prevent binding to the genomic DNA. For each gene tested, a standard curve was generated to determine the efciency of the reaction. PC enzyme activity assay Fluorometic assays were performed using 100 lM BOC-RVRR-AMC (Bachem) as a PC substrate. Equal volumes of the substrate, prepared in reaction buffer [25 mM Tris, 1 mM CaCl2, 0.5% (w/v) Brij-35, pH 9.0], and cell supernatants were incubated at 37C for 60 min. The uorescent AMC liberated by PC cleavage was measured at 380 nm excitation and 460 nm emission (Molloy et al. 1992; Ornatowski et al. 2007)

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224 Table 1 Sequences of the siRNA used for furin silencing in HEK-BMP2 cells

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siRNA Furin siRNA 1685 (F1) Furin siRNA 4142 (F2) Furin siRNA 516 (F3) Non-specic siRNA

siRNA sequence: sense/antisense 50 -GGCUCACCCUGUCCUAUAATT-30 50 -UUAUAGGACAGGGUGAGCCTT-30 50 -GACAUGAGAUAAUGUUAGATT-30 50 -UCUAACAUUAUCUCAUGUCTT-30 50 -GGCAAAGCGACGGACUAAATT-30 50 -UUUAGUCCGUCGCUUUGCCTT-30 50 -UUCUCCGAACGUGUCACGUTT-30 50 -ACGUGACACGUUCGGAGAATT-30

Table 2 Oligonucleotide primers for real-time RT-PCR analysis in CHO-BMP2 cells

Gene BMP2 FURIN GAPDH

Primer sequence: sense/antisense 50 -GCAGCCAGCCGAGCCAACACTG-30 50 -CTCCTCCGTGGGGATAGAA-30 50 -AGACCCCAAGTTTCCTCAGCAG-30 50 -AATGGAGACCACAATGCCGTGC-30 50 -TGTCCGTCGTGGATCTGAC-30 5 -CCTGCTTCACCACCTTCTTG-3

0 0

T (C) 60 60 55

Product (bp) 126 112 135

with a uorescence microplate reader. A standard curve using 7-amino-4-methyl-coumarin (AMC) was constructed. Data were normalized to cellular protein. For direct incubation, IND-1 at 10, 20, 40, 80, 160, 320 and 1000 lM was added to CHO cell lysates (107 cells lysed in 500 ll lysis reagent) and incubated at 37C for 30 min before the PC activity was measured. To measure the PC activity in cells cultured in media containing 20, 80, 500, and 1000 lM IND-1, cells were lysed after culture with IND-1 and the PC activity in the cell lysates was determined. Statistical analysis Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnetts multiple comparison post-hoc tests with statistical analysis software GraphPad PRISM v5 (GraphPad Software Inc., San Diego, USA). A P \ 0.05 was considered signicant.

its conditioned media was collected to determine the expression of the secreted recombinant human BMP-2 (rhBMP-2). Western blot analysis of the conditioned media under the non-reducing condition demonstrated an immunoreactive band at 36 kDa, co-migrating with a commercially available CHO-produced rhBMP-2 (Fig. 1a). The bioactivity of CHO secreted rhBMP-2 was determined using the C2C12 cell based assay. Murine myoblast C2C12 cells express very low levels of ALP in the absence of BMP. When BMP is present, the ALP activity in these cells increases proportionally (Katagiri et al. 1994). C2C12 bioassays were performed on the conditioned media of CHO cells cultured with IND-1 and a commercially available rhBMP-2 (as positive control). CHO secreted rhBMP2 induced ALP activity in a dose dependent manner (Fig. 1b). IND-1 signicantly increased rhBMP-2 yields To determine the efcacy of the polyarginine peptide IND-1 on enhancing rhBMP-2 production, CHO-BMP2 and HEK-BMP2 cells were cultured in media containing 0, 10, 20, 40 and 80 lM IND-1. After 24 h incubation, the amount of precursor BMP-2 (proBMP-2) and mature BMP-2 in the conditioned media was measured by ELISA. With 20, 40 and 80 lM IND-1, CHO-BMP2 had signicantly larger

Results Expression of active rhBMP-2 A stable CHO cell line (derived from the FlpIn system) expressing hBMP-2, CHO-BMP2, was cultivated and

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Fig. 1 a Anti-BMP-2 antibody probed western blot of commercially available CHO produced rhBMP-2 (as experiment control, CTRL) and conditioned medium sample from CHOBMP2 cells, under non-reducing conditions. In both lanes, BMP-2 dimers in appear as well-dened bands around 36 kDa. b Bioactivity of rhBMP-2 was measured by the induction of ALP activity in murine myogenic C2C12 cell line. The commercially available rhBMP-2 (120 ng/ml) was used as experiment control (CTRL). ALP activity is expressed as lmol ALP per h per mg total protein (lmol/h mg)

and proBMP-2 (Fig. 3b). To eliminate any differences in the cell culture other than the IND-1 treatment, a crossover was performed by interchanging the treatments between the groups on day 21. Following the crossover, IND-1 treated group continued to show higher mature and proBMP-2 yields than the untreated group up to 1.5- and 2-fold, respectively. These increases were lower than previously observed in the 24 h study. As a result, we further increased IND-1 from 20 to 60 lM on day 37, and consequently a larger increase in the BMP-2 yields was achieved: up to 2and 6-fold increases in mature and proBMP-2, respectively. Cell density and viability were measured at each media collection. The two groups of cells had similar densities throughout the study, regardless of the treatment received (Fig. 3c). Cells cultured with IND-1 also displayed similar cell viability, indicating that presence of IND-1 in the cell culture media had no adverse effect on the cells (Fig. 4). These results demonstrated that repeated incubation with IND-1 consistently improved mature and proBMP-2 yields in the CHO cell culture system without affecting cell growth and viability. IND-1 did not affect BMP2 or FURIN mRNA levels To investigate IND-10 s mechanism of action, BMP-2 and FURIN mRNA levels in CHO-BMP2 cells treated with or without 20 lM IND-1 was measured using quantitative RT-PCR. Furin is a prototypical member of the PC family and is believed to responsible for cleavage of BMP (Cui et al. 1998). Here we found that although rhBMP-2 protein level was elevated by up to 8-fold upon addition of IND-1 to the cell culture media (consistent with our previous results), mRNA levels of BMP2 and FURIN remained the same with or without IND-1 (Fig. 5). IND-1 inhibited PC activity in cell lysates, but not in cell culture Several studies have indicated that polyarginine peptides like IND-1 are potent inhibitors of furin (Cameron et al. 2000) and other members of the PC family (Fugere et al. 2007). CHO-BMP2 cell lysates were incubated with various concentrations of IND-1 and then assayed with the uorogenic peptide BOCRVRR-AMC containing the PC cleavage sequence-

amounts of mature BMP-2 and proBMP-2 secreted than the untreated group (P \ 0.01) (Fig. 2a). CHO cells cultured with 20 lM IND-1 demonstrated the largest increase in mature and proBMP-2 (6- and 37-fold, respectively). Likewise, HEK cells showed signicant increases in the secreted mature and precursor BMP-2 in all IND-1 treated groups (P \ 0.001) (Fig. 2b). HEK cells treated with 80 lM of IND-1 showed the greatest difference with 3- and 18-fold increase in the amount of mature and proBMP2 secreted, respectively. This study was repeated a minimum of three times for each cell line and reproducible results were obtained. To access IND-10 s long-term effect on BMP-2 yields and determine its efcacy as a media additive for recombinant protein production process, CHOBMP2 cells were cultured with IND-1 in perfusion cell culture asks over 53 days. The conditioned media from CHO cells incubated with IND-1 consistently measured higher amounts of mature BMP-2 (Fig. 3a)

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226 Fig. 2 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, respectively. Data are presented as mean SEM (n = 3) (**P \ 0.01, ***P \ 0.001)

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RVRR-. Upon cleavage, a uorescent product (AMC) was liberated, reecting PC activity. The data showed that the PC activity of CHO cell lysates was unchanged up to 80 lM of IND-1 treatment, the highest IND-1 concentration used in the cell culture media. At [80 lM IND-1, PC activity decreased signicantly (P \ 0.05) and was not measurable when the cell lysates were incubated with 1000 lM of IND1 (Fig. 6a). Next, intact cells were cultured in medium containing IND-1 for 24 h. After incubation, cell were lysed and the lysates were assayed for PC activity. None of the IND-1 treated group, up to 1000 lM, which is 50 times higher than required to affect the BMP-2 yields, showed any signicant change in PC activity compared to the untreated group (Fig. 6b). Furin knockdown did not affect rhBMP-2 yields To further investigate the relationship between intracellular furin expression and activity with BMP-2 yield, furin gene expression was transiently knocked down in HEK-BMP2 cells using three pairs of furinspecic siRNAs (F1, F2 and F3). Post-transfection, quantitative RT-PCR analysis on the cellular RNA level showed a reduction in furin mRNA level by more

than 80% (P \ 0.001, Fig. 7a) and ELISA analysis of the cell lysates showed signicantly reduced intracellular furin proteins (P \ 0.001, Fig. 7b) in the cells treated with furin-specic siRNAs. However, the activity assay showed no change in PC activity despite the reduction in the intracellular furin level. Furthermore, BMP-2 ELISA on the conditioned media demonstrated that cells treated with furin specic or non-specic siRNAs produced similar amount of BMP-2 (Fig. 7c). These results indicated that reduced intracellular furin level did not directly affect BMP-2 production in HEK-BMP2 cells.

Discussion Recombinant BMP-2 production in mammalian cell culture systems is relatively low compared to production of other recombinant human proteins of similar size. In this study, a polyarginine peptide IND-1 was used as a media additive and signicantly enhanced the yield of bioactive rhBMP-2. The stably transfected CHO-BMP2 cells secreted 36 kDa rhBMP-2 dimers, comparable in size to that reported in previous studies (Hillger et al. 2005; Israel et al. 1992). Cultivating

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Biotechnol Lett (2012) 34:221230 Fig. 3 Long-term effect of IND-1 on BMP-2 production in CHO cells. Two groups of CHO cells were cultivated in CELLline CL350 perfusion asks with or without IND-1 for a 53-day study. Cells were sub-cultured and the conditioned media were harvested from the cell compartment every 34 days. On day 5, one group of cells received the rst dose of 20 lM IND-1 and continued to be incubated with IND-1. On day 21, a crossover was performed, where the treatments of the two groups were switched (i.e. the group previously treated with IND-1, grew without IND-1 after the crossover). On day 37, IND-1 was increased to 60 lM. The amount of a proBMP-2 and b mature BMP-2 in the conditioned media was determined by pro- and mature BMP-2 ELISA, respectively. c At every media collection, the cell density of each group was determined

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Fig. 4 The viability of CHO cells cultured with (?IND-1) or without (-IND1) IND-1 was determined at every media collection over the 53-day study. Cell viability was determined by Trypan Blue exclusion method using Vi-CELL cell analyzer

CHO-BMP2 cells in the media containing IND-1 increased the amount of secreted pro- and mature rhBMP-2 by 40- and 6-fold, respectively, under

optimal culture conditions. In addition, this increase in BMP-2 yields upon IND-1 treatment was observed in two stably transfected cell lines, CHO-BMP2 and

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Fig. 5 Real-time RT-PCR analysis of the relative expression of BMP2 and FURIN in HEK-BMP2 cells cultured with or without IND-1 (left axis). Gene expression was analyzed with the iQ5 software (BioRad). Mature BMP-2 secreted by the cells was measured by BMP-2 ELISA (right axis). Data presented are mean values of two independent experiments

Fig. 6 PC activity was measured by monitoring the cleavage of uorogenic substrate boc-RVRR-amc. PC activity is expressed as lmol AMC released from boc-RVRR-amc per h per g total protein (lmol/h.g). a PC activity in the CHO-BMP2 cell lysates after direct incubation with IND-1. b PC activity measured in cell lysates of CHO-BMP2 cells cultured with cell culture media containing IND-1 for 24 h. Data are presented as mean SEM (n = 3) (*P \ 0.05, **P \ 0.01)

HEK-BMP2. Furthermore, repeated use of IND-1 led to consistent increases in BMP-2 yields over 53 days without affecting cell growth or viability at concentrations used. These data demonstrated that IND-1 could be used as a media additive to enhance rhBMP-2 production in mammalian cell expression systems. We undertook a series of experiments to investigate the mechanism by which IND-1 enhanced the yields of rhBMP-2. Many groups have also reported internalization of polyarginine peptides by CHO cells (Fuchs and Raines 2004) and other cell types (Heng et al. 2010; Hess et al. 2007; Kibler et al. 2004). We have shown that uorescent-labeled IND-1 peptides were taken up by the CHO cells (SAF Peel et al. unpublished data). Since the gene expression of BMP2 and FURIN in cells cultured with IND-1 did not change, we examined other post-transcriptional processes, further down the BMP-2 biosynthetic pathway. Polyarginines are known inhibitor for furin (Cameron et al. 2000; Kacprzak et al. 2004) and other members of the PC family (Fugere et al. 2007). We speculated that the increase in BMP-2 yields might result from IND-10 s inhibition on PC activity. Surprisingly, the overall PC enzymatic activity in the CHO-BMP2 cells following IND-1 incubation was not reduced, although direct incubation of CHO cell lysates with IND-1 at high doses (C160 lM) demonstrated inhibition of overall PC activity. These results suggest that IND-1 at the concentration used was not sufcient to inhibit PC activity. In addition, after furin expression in CHO-BMP2 cells was knocked down by RNA silencing, the BMP-2 secreted by these cells remained unchanged, indicating that the majority of PC activity in HEK cells was contributed by PCs other than furin, furthermore suggests that furin may not be the main PC in processing BMP-2 in HEK cells. When CHO-BMP2 cells were incubated with another synthetic PC inhibitor decanoyl-RVKR-chloromethylketone (CMK) (Angliker et al. 1993; Stieneke-Grober et al. 1992) at similar concentrations to IND-1, the amount of proBMP-2 secreted increased signicantly (P \ 0.001). However, the amount of mature BMP-2 secreted by CHO cells decreased signicantly (Supplementary material Fig. 1, P \ 0.001), unlike with IND-1 treated cells, where the mature BMP-2 yield increased notably. Hence, we conclude that IND1 at the concentrations used did not increase BMP-2 yields by inhibiting cellular PC activity.

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Biotechnol Lett (2012) 34:221230 Fig. 7 The effects of three furin-specic siRNA pairs on a FURIN gene expression, b intracellular furin level, c PC activity and d mature rhBMP-2 yield, 48 h post-transfection. Cells treated with non-specic siRNA were used as experiment control. Data in a and b are presented as percentage of furin mRNA and furin protein (respectively) remaining after the knockdown, for which 100% corresponds to non-furin siRNA treated cells. Data are presented as mean SD (n = 2) (***P \ 0.001)

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L-Arginine has been widely used as an enhancer of protein renaturation. It was shown to suppress protein aggregation by increasing the solubility of unfolded protein species and intermediates during oxidative refolding (Reddy et al. 2005). Schaffner et al. demonstrated the addition of 0.4 M L-arginine to the culture medium increased the yield of recombinant plasminogen activator in an E. coli expression system. The results on culturing CHO-BMP2 cells with D-arginine (equivalent to 80 lM IND-1) demonstrated no increase in secreted rhBMP-2 (Supplementary material Fig. 2). In addition, a set of polyarginine peptides consisting of seven positively charged D-arginines (7R), but with different net charges were created by inserting negatively charged aspartic acids (D) at the ends or throughout the peptide. When CHOBMP2 cells were incubated with these 7R peptides, only the highly positive charged 7R peptide (with a net charge of ?5) elicited an increase in BMP-2 yield (Supplementary material Fig. 3). However, the increase in BMP-2 was not as high as when CHO cells were incubated IND-1 (6- vs. 2-fold), which has nine D-arginines. These data suggest that the overall charge of the polyarginine peptide affects its ability to alter BMP-2 yield. To understand its role in enhancing rhBMP-2 production, future studies should explore the biophysical property of IND-1, in addition to its biological roles.

The low yield of recombinant human BMP-2 in mammalian cell culture systems has led to investigations to enhance its production. By adding the IND-1 peptide to the cell culture medium, a signicant increase of bioactive BMP-2 proteins was produced by CHO and HEK cell expression systems and was sustainable for long-term production. IND-1 can be a useful cell culture media additive to enhance recombinant BMP-2 yields in mammalian cell culture systems.
Acknowledgment This work was supported in part by Oral Surgery Foundation of Canada and McEwen Research Fund (#199-03-1570). A.J.Z. is a recipient of a Natural Sciences and Engineering Council Post-Graduate Scholarship (NSERC-PGS) scholarship. We are grateful to Dr. Michelle Visser for careful scrutiny of several drafts of this manuscript.

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