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Biotechnology Letters

, Volume 37, Issue 9, pp 1773–1781 | Cite as

Functional expression of the human coagulation factor IX using heterologous signal peptide and propeptide sequences in mammalian cell line

  • Shohreh Khorshidi
  • Alireza Zomorodipour
  • Mehrdad Behmanesh
  • Jafar Vatandoost
  • Mettine H. A. Bos
Original Research Paper
First Online:
Received:
Accepted:

Abstract

Objective

To study the functions of pre-pro leader peptides of the human and porcine prothrombins on the human FIX (hFIX) expression.

Results

In silico analysis predicted higher secretion efficiencies for the prothrombins-derived signal peptides, in comparison with the native hFIX signal peptide. Replacements of the hFIX pre-pro sequence with those of the two prothrombins, led to increased levels of transcription of the chimeric transgenes, as compared to the native clone. This was in consistent with the lower minimum free energies, calculated for the recombinant transcripts, based on their secondary structures. Evaluation of secretion efficiency revealed that the highest and lowest FIX secretions belong to signal peptides derived from porcine’ prothrombin and hFIX, respectively. Coagulation activities of the FIX expressed from chimeric variants could be increased up to tenfold, relative to the native clone.

Conclusion

The feasibility of a leader-peptide replacement for the improvement of both transcription and post-transcriptional processes is described that can be relevant for production the vitamin-K dependent proteins.

Keywords

γ-Carboxylation Human coagulation factor IX Human prothrombin Porcine prothrombin Propeptide Signal peptide 

Introduction

Factor IX (FIX) is a vitamin K-dependent plasma protein that participates in the intrinsic pathway of blood coagulation. It is activated by either factor XIa or by tissue factor/factor VIIa complex and followed by converting factor X to its active form in the presence of Ca2+ ions, phospholipids, and factor VIIIa (Jackson and Nemerson 1980). FIX, expressed as a prepropolypeptide precursor, is composed of a signal peptide and a propeptide connected to the mature protein N-terminus (Mannucci 2008). FIX undergoes numerous post translational modifications (PTMs), among them γ-carboxylation has a significant impact on its biological activity (Walsh 2010; Orlova et al. 2012). γ-Carboxylation is mediated by two essential enzymes: a VKD γ-carboxylase (γC), which requires reduced vitamin K as a cofactor, and vitamin K 2,3-epoxide reductase (VKOR), which produces the cofactor (Wallin and Hutson 2004). γ-Carboxylase recognizes its substrate through its propeptide which is removed by an endoprotease, called paired basic amino acid cleaving enzyme (PACE/Furin) (Pan and Price 1985; Jorgensen et al. 1987).

Mammalian cells are generally considered as the most suitable host for production of rhFIX, because of their capability of PTMs on the proteins produced (Wajih et al. 2005). The competence of a protein being processed and translocated through the endoplasmic reticulum (ER) membrane is highly dependent on the corresponding signal peptide which is cleaved during secretion of the protein in ER (Zhang et al. 2005). Functional efficiency of a signal peptide is directly related to its hydrophobicity (Tomilo et al. 1994). In spite of conservation of the structure of signal sequences and the operation of the secretory machineries, not all signal peptides are interchangeable among different proteins and organisms (Tessier et al. 1991), because additional features might also contribute to the secretion efficiency (Galliciotti et al. 2001). Propeptide, which plays critically important function in the VKD protein γ-carboxylation, is essential for substrate recognition by γ-carboxylase (Pan and Price 1985). Due to its position, immediately next to signal peptide, it might also impose great impact on secretion of the linked protein as well. Propeptides behave differently, when their affinities towards γ -carboxylase is considered.

The affinity of propeptides for the γ-carboxylase varies over 2 logs, with the lowest affinity for prothrombin that can explain a high efficient γ-carboxylation of the human prothrombin (hProt) (Stanley et al. 1999; Blostein et al. 2008). A primary comparison of the nucleotide and amino acid sequences of prothrombin of porcine, canine, cat and human, demonstrated respective similarities of 86.2 and 83 %, between those of human and porcine. Besides, previous studies showed that the porcine’s coagulation factors can affect the intrinsic and extrinsic coagulation pathways in human (Chen et al. 2007). So, replacement of the hFIX prepro-leader peptide with those of the hProt and pProt was thought to be worth examining. Exchanging the propeptide of the hFX, expressed in the HEK293T cell line, with that of the hProt substantially enhanced γ-carboxylation (Camire et al. 2000). In this work, expression of recombinant hFIXs derived from two chimeric clones, equipped with either of the hProt and pProt leader peptides, in comparison with that one with a native leader peptide, in transiently transfected mammalian cells, was studied.

Materials and methods

Signal peptide predictions

The signal peptides’ efficiencies, for their biological activities in a eukaryotic cell, were tested with the help of the neural network-based SignalP (Nielsen et al. 1997), and PrediSi programs (Hiller et al. 2004). TopPred program (Claros and von Heijne 1995), a topology prediction of membrane proteins, was used for hydrophobicity analysis of the signal peptides. Hydropathy plots of the amino acids were made using hydropathy values for amino acids taken from Eisenberg et al. (1982).

Prediction of mRNA secondary structures

RNA structures of a sequence covering the 5′-end of mRNAs including; translation start codon, signal sequence and part of coding region of the mature protein N-terminus were analysed using the algorithm of favourite thermodynamic by Vienna RNA secondary structure prediction program at http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi (Hofacker 2003).

Construction of recombinant plasmids

Using a previously made plasmid (Vatandoost et al. 2012), as a backbone, two recombinant plasmids equipped with either the pProt or hProt pre-pro leader sequences, instead of the native hFIX pre-pro, were constructed. For the construction of the first plasmid, the pProt pre-pro coding sequence was included in a 486 bp KpnI/EcoRV fragment, covering a section of the hFIX cDNA, corresponding to the mature protein N-terminal. The designed fragment was synthesized by ShineGene Company, in the puc57 plasmid. Following the KpnI/EcoRV digestion the pProt pre-pro coding sequence was used to substitute the native hFIX pre-pro sequence in the native hFIX expressing plasmid, which resulted in a plasmid, named as pProt-hFIX. For the construction of the second plasmid, a 129 bp corresponding to the coding sequence of the hProt pre-pro leader peptide fused to the hFIX mature protein, was amplified using oligonucleotides Hprot-KpnI and hFIX-XhoI as forward and reverse primers, respectively (Supplementary Table 1), which resulted in a plasmid labelled as hProt-hFIX. In all constructs a human Kozak sequence was engineered prior to the corresponding start codon. The identity of the recombinant plasmids was confirmed, by employing restriction digestion followed by nucleotide sequencing.

Cell culture and transfection optimization

HEK293T (Pasteur Institute, Iran) cells were maintained in DMEM:Ham’s F12 (1:1) medium supplemented with 4 mM l-glutamine, 10 % (v/v) fetal bovine serum (FBS), in addition to 100 U penicillin/ml and 100 µg streptomycin/ml and grown at 37 °C with 5 % CO2. One day prior to transfection, 2.5 × 105 cells were seeded in 2.5 ml of medium in six-well plates, with 2 μg plasmid DNA, 6 µl X-tremeGENE9 DNA reagent (Roche) according to the manufacturer’s instruction. Five h after transfection, fresh rich medium [containing 6 μg vitamin K1/ml and 10 % (v/v) FBS] was added to the cells. During 3 days of post-transfection, the cultured media were harvested for transient expression analysis. The potential matrix effect was examined by using both lysate and media from non-transfected cells in parallel with the main expression assays.

Immunoassay of the expressed hFIX

The quantitative measurement of the expressed hFIX in the culture media was performed through standard enzyme-linked immunosorbent assay (ELISA) (Asserachrom hFIX::Ag) with specific Rabbit anti-hFIX antibodies coupled with peroxidase according to the kit instruction and were measured based on the standard curve and reported in ng/ml.

Analysis of intracellular accumulation of hFIX

The conditioned cultured cells were precipitated in ice-cold lysis buffer (EDTA, 50 mM Tris/HCl, 100 mM NaCl, 1 % Triton X-100) containing an antiprotease mix (Roche) for 15 min, followed by centrifugation at 12,000×g for 15 min at 4 °C. The hFIX content of the supernatant was measured by ELISA and stated in ng/ml.

hFIX coagulation activity assay

FIX activity in the cultured media was measured by a chromogenic assay according to the protocol (Biophen factor IX kit). We used the human normal citrated plasma as a standard sample. The cultured medium of non-transfected cells was used as a negative control.

hFIX γ-carboxylation assay

Precipitation of γ-carboxylated FIX was performed based a modified method described by Patrizia et al. (1987). Briefly, a mixture of 2 % (w/v) sodium citrate (20 μl) and 1 M BaCl2 (50 mg) plus 1 ml medium was incubated at 4 °C, mixing gently, for 1 h, followed by centrifugation at 300×g for 5 min. The supernatant was collected for further analysis of unabsorbed FIX and the precipitant (γ-carboxylated FIX) was washed with ice-cold 5 mM BaC12 and recentrifuged at 300×g for 5 min. The precipitant was resuspended in 0.1 M sodium citrate (500 μl) and adjusted to 10 % (w/v) (NH4)2SO4 to dissolve the absorbed FIX. In order to remove the undesired barium sulphate pellets, the mixture was incubated for 30 min at 4 °C and followed by a centrifugation at 300×g for 5 min. The amount of the FIX in the supernatant was determined by ELISA.

RNA analysis

Using Qiazol lysis reagent, total RNAs were extracted from the cells and were reverse transcribed, using random primers and revert Aid M-MuLV. The obtained cDNAs were used as a template for subsequent PCR experiments with two hFIX-specific oligonucleotides HFIX-RTR1 and HFIX-RTF1 as forward and reverse primers, respectively (Supplementary Table 1). A 113 bp fragment of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was amplified as an internal control using oligonucleotides Hu-GAPDH-F and Hu-GAPDH-R as forward and reverse primers, respectively (Supplementary Table 1). Real-time PCR was performed using SYBR green method and premix Amplicon kit on an ABI 7500 real-time PCR system.

Statistical analysis

Coagulation test and ELISA were carried out in triplicates and the generated data were presented as the mean ± SD. The data collected were subjected to analysis of variance test. The means were compared using one-way ANOVA test. A p value of less than 0.05 was considered as significant difference. All statistical analyses were carried out with GraphPad Prism version 5 (Graph-Pad Software Inc., San Diego, CA, USA).

Results

Computational evaluation of the signal peptides

Using two neural network-based programs, SignalP and PrediSi, processing efficiencies of the hFIX, hProt and pProt were predicted that signal peptides fused to their cognate propetides in a eukaryotic host (Table 1). The two exogenous signal peptides were predicted to be cleaved precisely on their natural cleavage positions, with relatively high scores. The highest efficiency of the hFIX secretion was expected when the pProt leader sequence was used. Hence, the respective potentials of the signal peptides for secretion of hFIX were; pProt > hProt > hFIX. For a signal peptide, to be recognized by Signal Recognition Particle (SRP), total hydrophobicity is an important factor rather than the length of H-region (Hatsuzawa et al. 1997; Stern et al. 2007). Our findings, based on TopPred program and performed on the amino acid sequences of the three signal peptides (see Supplementary Fig. 1), showed that the total hydrophobicity of the pProt signal peptide is higher than those of the hProt and hFIX. These results were confirmed by the hydropathy values obtained from Eisenberg et al. (1982) (Fig. 1).
Table 1

The first 70 amino acids of N-terminus of each preproprotein were considered for signal peptide predictions

Software

SignalP

Predisi

Score

Prediction

Score

Prediction

hFIX

0.672

SP

0.5324

SP

pProt

0.924

SP

0.9090

SP

hProt

0.739

SP

0.7033

SP

In the case of SignalP output, the cleavage sites are predicted with the combined peaks from C-score and Y-score on a single site. In the case of the Predisi output, Score: combined cleavage site score

Fig. 1

Hydropathy plots of the amino acids in the signal peptides. a Native hFIX, b pProt-hFIX (pProt) and c hProt-hFIX (hProt). Plots were made using hydropathy values for amino acids taken from Eisenberg et al. (1982)

Secondary structure prediction of the hFIX transcripts

The secondary structure of mRNA has a major impact on protein biosynthesis due to its potential for limiting the movement of ribosomes (Gaspar et al. 2013). For each signal peptide, the 5′-end of its corresponding mRNAs including part of coding region of the mature protein N-terminus was considered for prediction of its potential secondary structure. In order to evaluate the strengths of the mRNAs, their minimum free energies (MFE (were calculated. As the results showed, the MFEs for the pProt-hFIX and hProt-hFIX (with a theoretical ∆G values of −389.1 and −390.5 kcal/mol, respectively) were higher than that of the native hFIX (−775.68 kcal/mol). Therefore, the chimeric transcripts were expected to be accessible more easily to ribosomes. This might lead to higher translation rates in comparison with that of the native hFIX transgene. In subsequent steps, we investigated the efficiency of the three signal peptides, in combination with their linked propeptide, experimentally, for the secretion of hFIX in cultured mammalian cells.

Transient expression analysis

Transfection of the cells with the recombinant FIX expressing plasmids was confirmed by visualizing of their corresponding transcripts. Indeed, the electrophoresis pattern of the RT-PCR products generated by specific primers indicated in the presence of the hFIX mRNAs within the RNA-pools of each of the transfected cells (Fig. 2). To investigate the variation of FIX expression due to of the prepro-leader sequence modifications, a real-time quantitative PCR, based on comparative ΔΔCT method (Livak and Schmittgen 2001), was performed. With this aim, the hFIX transcripts obtained from the three variant transgenes were compared to that of the native clone. Normalized to the endogenous levels of the transcripts in non-transfected cells, the expression levels of the FIX, mediated by the three pre-pro leader sequences were evaluated. More than 16- and 6-fold increase in the transcript level of the hFIX occurred as a result of the presence of the pProt and hProt leader peptides, respectively, with respect to the clone with the native hFIX pre-pro sequence (see Supplementary Fig. 2). For each of the transfectants, during 72 h post-transfection the secreted hFIX was estimated based on its antigenicity (Fig. 3). The amount of FIX in the cultured media, taken from the cell pool with the native hFIX, was higher than those with prothrombin prepro leader peptides. In all cases, protein levels found in the culture medium were significantly higher than those detected in cell lysates, suggesting for efficient secretions of the mature proteins for the three transgenes.
Fig. 2

PCR products amplified from the reverse transcribed total RNA, extracted from transfected cells at 24, 48 and 72 h. Lanes 1 DNA size marker (100 bp), lanes 2–4 pProt-hFIX (pProt), lanes 5–7 hProt-hFIX (hProt), lanes 8–10: hFIX, lanes 11 lysed non-transfected cells, lanes 12 negative control

Fig. 3

Evaluation of transient expression of the hFIX in HEK293T cells at various post-transfection times, based on ELISA

Evaluation of the hFIX secretion efficiency

Secretion efficiency of a particular protein is defined as the secreted fraction in the medium divided by its total (secreted and cellular) quantity. The secretion efficiency of FIX for the cells transfected with the three recombinant plasmids, varied between 86 and 91 % (for pProt-hFIX), 83–89 % (for hProt- hFIX) and 83–86 % (for native hFIX) during 24–72 h post-transfection (Fig. 4). At 24 h post-transfection, no significant differences were observed among the studied clones. However, during next 48 h incubation, the secretion efficiency of the product of pProt- hFIX increased and remained more or less constant during next 24 h. Thus, the highest secretion efficiency was obtained from pProt-hFIX at 72 h post-transfection.
Fig. 4

Assessment of the hFIX secretion efficiency from HEK293T. Asterisks indicate samples that are significantly different (*p < 0.05; **p < 0.01; ***p < 0.001) compared to other samples, using analysis of variance

Activity measurements of the expressed hFIX

The clotting activity of FIX was measured in conditioned media from transfected cells at different time points. Our results indicated that the biological activities of the expressed hFIXs, reached up to 180 and 160 mU/ml at 24 h post-transfection for pProt-hFIX and hProt-hFIX constructs, respectively, which are significantly higher than that of the FIX expressed from the native clone (Fig. 5). A specific feature of a fully γ-carboxylated VKD protein is to be absorbed to barium citrate through its γ-carboxylated Glu residues (Jallat et al. 1990). Analysis of the FIXs recovered from barium citrate precipitants, showed a range of 30–36 % of the adsorbed FIX, secreted by three recombinant cells, with no significant differences. The barium citrate-precipitated FIX is most likely composed of the fully γ-carboxylated FIX. Therefore, the non-precipitated portions (64–70 %) are thought to be partially or not γ-carboxylated at all.
Fig. 5

Evaluation of the expressed hFIX activity, based on chromogenic assay. Asterisks indicate samples that are significantly different (p < 0. 0001) compared to other samples, using analysis of variance

Discussion

In either of the applications in which secretion of the transgene product is aimed, accumulation of the expressed protein within the secretory compartments, is a major hindrance (Rehemtulla et al. 1993). Previously, we observed the accumulation of the expressed hFIX inside the recombinant mammalian cells, when its native signal peptide was applied (Sam et al. 2010). Among different parameters involved in secretion efficiency of a protein, signal peptide plays important function and has impact on the regulation of gene expression as well (Stern et al. 2007; Soejima et al. 2013). Therefore, achieving an efficient secretion of the protein of interest is a major concern and choice of suitable signal peptide might be helpful. Consistent with our in silico predictions, improvement of FIX secretion, when joined to either of the examined prepro sequences, was demonstrated in the human cell line. Based on these results, with the application of a heterologous signal peptide, a mammalian cell can produces FIX with secretion efficiency up to 91 %, with estimated 125 ng/ml per106 cells. For the cells with either pProt or hProt pre-pro sequences, the trapped FIX within the cellular space decreased as the cultivations continued, indicating in the improvement of FIX secretion. Whereas, this was not the case for the native hFIX expressing cells. The higher hydrophobicities of the pProt and hProt than that of the hFIX which was demonstrated in our in silico analysis and the relatively higher secretion efficiencies of the two prothrombin derived signal peptides is in agreement with the results reported by Hatsuzawa et al, (1997) who demonstrated that, for a signal peptide to be recognized by SRP, total hydrophobicity is an important factor rather than the length of H-region. Bird et al, (1990) suggested that the efficiency of a protein translocation is related to hydrophobicity degree of the corresponding signal peptide. Zhang et al. (2005) observed up to 3.5 fold enhancement in secretion level of IL-2 through increasing the hydrophobicity of the h-region and the basicity of the N-region of signal peptide.

In a more related work to present study, replacement of the native signal sequence with that of the Gaussia princep luciferase signal peptide had a major impact on the synthesis and secretion of the protein (Knappskog et al. 2007). A common feature of most secreted proteins are post-translational modifications. PTMs, which are required for their biological activities (Hallgren et al. 2002). The coagulation activities of rhFIX, expressed by the examined constructs, indicated their occurrence of γ-carboxylation. Our result is in consistent with the results reported by Camire et al. (2000), who showed the ability of human γ-carboxylase to recognize the propeptide of the hProt as substrate. Therefore, either of the pProt and hProt propeptides that bind to γ-carboxylase with a low affinity seems to be potent substrates for γ-carboxylation. A sharp decrease in the content of the biologically active FIX after 24 h post-transfection in the cases of the two chimeric transgenes, suggested a probable decline of the γ-carboxylated protein during the examination time. Therefore the opposing results obtained from coagulation test and barium citrate precipitation on the same samples could be explained by self-degradation of the expressed protein that to be elucidated.

Our quantitative RT-PCR results showed that the mRNA level can be altered dramatically after the leader sequence replacement. This result is in contrast to the results obtained by Tröβe et al. (2007) and Knappskog et al. (2007) who demonstrated that, the mRNA levels of the target gene is largely unaffected in mammalian cells, when a heterologous signal peptides was used. The transcripts’ MFE values predicted higher translation rates for the transgenes with either of the examined prothrombin prepro leader sequences, whereas, the highest expression level was obtained from the native transgene. This is also in contrast to our quantitative results at transcription levels. This unprecedented result can be explained by lower translation efficiencies of the two heterologous leader-peptides that could possibly be improved by optimization of their codon profiles.

In conclusion, our findings confirmed that the coding region of a heterologous leader peptide can effect both transcription and post-transcriptional processes. Moreover, features such as mRNA secondary structure, signal peptide hydrophobicity and processing efficiency and its combination with propeptide, play important roles in the expression efficiency of the target protein. The presented results are from transient expression state, which indicates in possibility of a leader peptide replacement approach for improvement of the secretion efficiency of a protein of interest. A considerable number of cells within the examined cell-pools were not transfected that could effect on the final expression output. Therefore, next step to this work is to examine the constructed plasmids in stable transfection state, in which further optimization of the culture media as well as growth conditions would be necessary to achieve an efficient expression of hFIX.

Notes

Acknowledgments

This work was performed as part of the requirement for the fulfilment of the degree of PhD in Molecular Genetics of S. Khorshidi at Tarbiat Modares University (TMU) and the National Institute of Genetic Engineering and Biotechnology (NIGEB) of Iran and financially supported in part by a grant (No. 419) at NIGEB.

Supporting information

Supplementary Table 1—List of the oligonucleotides, used for the constructions of the hFIX expression cassettes and RT-PCR and real-time pCR.

Supplementary Figure 1—Hydropathy plots of the amino acids in the signal peptides by using TopPred online program.

Supplementary Figure 2—QRT-PCR analysis on hFIX transcripts produced by the three variant transgenes.

Supplementary material

10529_2015_1868_MOESM1_ESM.docx (171 kb)
Supplementary material 1 (DOCX 172 kb)
10529_2015_1868_MOESM2_ESM.docx (11 kb)
Supplementary material 2 (DOCX 11 kb)

References

  1. Bird P, Gething M, Sambrook J (1990) The functional efficiency of a mammalian signal peptide is directly related to its hydrophobicity. J Biol Chem 265:8420–8425PubMedGoogle Scholar
  2. Blostein M, Cuerquis J, Landry S, Galipeau J (2008) The carboxylation efficiency of the vitamin K-dependent clotting factors: studies with factor IX. Haemophilia 14:1063–1068CrossRefPubMedGoogle Scholar
  3. Camire RM, Larson PJ, Stafford DW, High KA (2000) Enhanced γ-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. Biochemistry 39:14322–14329CrossRefPubMedGoogle Scholar
  4. Chen Y, Tan W, Lu X, Lu Y, Qin S, Li S, Zeng Y, Bu H, Li Y, Cheng J (2007) Full-length cDNA cloning and protein three-dimensional structure modeling of porcine prothrombin. Blood Cell Mol Dis 38:93–99CrossRefGoogle Scholar
  5. Claros MG, von Heijne G (1995) TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10:685–686Google Scholar
  6. Eisenberg D, Weiss RM, Terwilliger TC, Wilcox W (1982) Hydrophobic moments and protein structure. Faraday Symp Chem Soc 17:109–120CrossRefGoogle Scholar
  7. Galeffi P, Brownlee GG (1987) The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid-4. Nucleic Acids Res 15(22):9505–9513PubMedCentralCrossRefPubMedGoogle Scholar
  8. Galliciotti G, Schneider H, Wyder L, Vitaliti A, Wittmer M, Ajmo M, Klemenz R (2001) Signal-sequence trap in mammalian and yeast cells: a comparison. J Membr Biol 183:175–182CrossRefPubMedGoogle Scholar
  9. Gaspar P, Moura G, Santos MA, Oliveira JL (2013) mRNA secondary structure optimization using a correlated stem–loop prediction. Nucleic Acid Res 41:e73PubMedCentralCrossRefPubMedGoogle Scholar
  10. Hallgren KW, Hommema EL, McNally BA, Berkner KL (2002) Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: implications for the release of vitamin K-dependent proteins. Biochemistry 41:15045–15055CrossRefPubMedGoogle Scholar
  11. Hatsuzawa K, Tagaya M, Mizushima S (1997) The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane. J Biochem 121:270–277CrossRefPubMedGoogle Scholar
  12. Hiller, K., A. Grote, M. Scheer, R. Munch and D. Jahn (2004). PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acid Res 32(Web Server issue): W375–W379PubMedCentralCrossRefPubMedGoogle Scholar
  13. Hofacker IL (2003) Vienna RNA secondary structure server. Nucleic Acid Res 31:3429–3431PubMedCentralCrossRefPubMedGoogle Scholar
  14. Jackson CM, Nemerson Y (1980) Blood coagulation. Ann Rev Biochem 49:765–811CrossRefPubMedGoogle Scholar
  15. Jallat S, Perraud F, Dalemans W, Balland A, Dieterle A, Faure T, Meulien P, Pavirani A (1990) Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines. EMBO J 9:3295–3301PubMedCentralPubMedGoogle Scholar
  16. Jorgensen MJ, Cantor AB, Furie BC, Brown CL, Shoemaker CB, Furie B (1987) Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX. Cell 48:185–191CrossRefPubMedGoogle Scholar
  17. Knappskog S, Ravneberg H, Gjerdrum C, Tröβe C, Stern B, Pryme IF (2007) The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide. J Biotechnol 128:705–715CrossRefPubMedGoogle Scholar
  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  19. Mannucci PM (2008) Back to the future: a recent history of haemophilia treatment. Haemophilia 14(Suppl 3):10–18CrossRefPubMedGoogle Scholar
  20. Nielsen H, Engelbrecht J, Brunak S, Heijne GV (1997) A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8:581–599CrossRefPubMedGoogle Scholar
  21. Orlova NA, Kovnir SV, Vorobiev II, Gabibov AG (2012) Coagulation factor IX for hemophilia B therapy. Acta Nat 4(2):62–73Google Scholar
  22. Pan LC, Price PA (1985) The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors. Proc Nat Acad Sci USA 82:6109–6113PubMedCentralCrossRefPubMedGoogle Scholar
  23. Rehemtulla A, Roth DA, Wasley LC, Kuliopulos A, Walsh CT, Furie B, Furie BC, Kaufman RJ (1993) In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells. Proc Natl Acad Sci 90(10):4611–4615PubMedCentralCrossRefPubMedGoogle Scholar
  24. Sam MR, Zomorodipour A, Shokrgozar MA, Ataei F, Haddad-Mashadrizeh A, Amanzadeh A (2010) Enhancement of the human factor IX expression, mediated by an intron derived fragment from the rat aldolase B gene in cultured hepatoma cells. Biotechnol Lett 32:1385–1392CrossRefPubMedGoogle Scholar
  25. Soejima Y, Lee JM, Nagata Y, Mon H, Iiyama K, Kitano H, Matsuyama M, Kusakabe T (2013) Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system. Centr Eur J Biol 8(1):1–7Google Scholar
  26. Stanley TB, Jin D-Y, Lin P-J, Stafford DW (1999) The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase. J Biol Chem 274:16940–16944CrossRefPubMedGoogle Scholar
  27. Stern B, Olsen LC, Tröße C, Ravneberg H, Pryme IF (2007) Improving mammalian cell factories: the selection of signal peptide has a major impact on recombinant protein synthesis and secretion in mammalian cells. Trend Cell Mol Biol 2:1–17Google Scholar
  28. Tessier DC, Thomas DY, Khouri HE, Laliberié F, Vernet T (1991) Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene 98:177–183CrossRefPubMedGoogle Scholar
  29. Tomilo M, Wilkinson KS, Ryan P (1994) Can a signal sequence become too hydrophobic? J Biol Chem 269:32016–32021PubMedGoogle Scholar
  30. Tröβe C, Ravneberg H, Stern B, Pryme I (2007) Vectors encoding seven oikosin signal peptides transfected into CHO cells differ greatly in mediating Gaussia luciferase and human endostatin production although mRNA levels are largely unaffected. Gene Regul Syst Biol 1:303–312Google Scholar
  31. Vatandoost J, Zomorodipour A, Sadeghizadeh M, Aliyari R, Bos MH, Ataei F (2012) Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme. Biotechnol Prog 28:45–51CrossRefPubMedGoogle Scholar
  32. Wajih N, Hutson SM, Owen J, Wallin R (2005) Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle. J Biol Chem 280:31603–31607CrossRefPubMedGoogle Scholar
  33. Wallin R, Hutson SM (2004) Warfarin and the vitamin K-dependent gamma-carboxylation system. Trend Mol Med 10:299–302CrossRefGoogle Scholar
  34. Walsh G (2010) Post-translational modifications of protein biopharmaceuticals. Drug Disc Today 15:773–780CrossRefGoogle Scholar
  35. Zhang L, Leng Q, Mixson AJ (2005) Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo. J Gene Med 7:354–365CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Shohreh Khorshidi
    • 1
  • Alireza Zomorodipour
    • 2
  • Mehrdad Behmanesh
    • 1
  • Jafar Vatandoost
    • 3
  • Mettine H. A. Bos
    • 4
  1. 1.Department of Molecular Genetics, Faculty of Biological SciencesTarbiat Modares UniversityTehranIran
  2. 2.Department of Molecular GeneticsNational Institute of Genetic Engineering and BiotechnologyTehranIran
  3. 3.Department of Basic SciencesSabzevar University of Medical SciencesSabzevarIran
  4. 4.Department of Thrombosis and HaemostasisLeiden University Medical CentreLeidenThe Netherlands

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