Expression of recombinant human mutant granulocyte colony ...

World J Microbiol Biotechnol (2012) 28:2593–2600 DOI 10.1007/s11274-012-1068-4

ORIGINAL PAPER

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli F. R. Gomes • A. C. Maluenda • J. O. Ta´pias • F. L. S. Oliveira • L. C. Sa´-Rocha • E. Carvalho P. L. Ho

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Received: 26 January 2012 / Accepted: 20 April 2012 / Published online: 1 May 2012 Ó Springer Science+Business Media B.V. 2012

Abstract The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this

Electronic supplementary material The online version of this article (doi:10.1007/s11274-012-1068-4) contains supplementary material, which is available to authorized users. F. R. Gomes E. Carvalho (&) P. L. Ho (&) Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, Saõ Paulo, SP 05503-900, Brazil e-mail: [email protected] P. L. Ho e-mail: [email protected] A. C. Maluenda J. O. Ta´pias F. L. S. Oliveira CIALLYX Laborato´rios & Consultoria, Saõ Paulo, Brazil L. C. Sa´-Rocha Laborato´rio de Neuroimunologia, Departamento de Patologia, Faculdade de Medicina Veterina´ria e Zootecnia da Universidade de Saõ Paulo, Saõ Paulo, Brazil

recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market. Keywords Recombinant human granulocyte colony stimulating factor G-CSF Nartograstim Expression system High yields Escherichia coli

Introduction Neutrophils, the predominant white cells in blood, are considered the first line of innate defense against bacteria. They need to be constantly replaced in the organism, since they circulate in blood for 8–12 h and can be functional for only 2–5 days after migration into the tissues (Li et al. 2002). Its proliferation is mainly controlled by the granulocyte-colony stimulating factor (G-CSF), one of the growth stimulating molecules known as colony stimulating factors (CSF), which constitute a group of proteins that control the proliferation and differentiation of hematopoietic cells. More precisely, the human G-CSF (hG-CSF) stimulates and regulates the proliferation, survival and differentiation of neutrophils (Clark and Kamen 1987). Besides, this protein also contributes to other functions and biological processes. The hG-CSF regulates the level of neutrophils circulating systemically (Welte et al. 1996), and also stimulates their bactericidal activity, chemotaxis and phagocytosis (Weisbart and Golde 1989). At a minor extent, hG-CSF also acts on other leukocytes, enhancing the number of agranulocyte white cells (lymphocytes and monocytes) in the circulating systemic blood (Sica et al. 1996), and on non-hematopoietic cells by, for instance, stimulating the proliferation and migration of vascular endothelial cells (Barreda et al. 2004). Finally, hG-CSF

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promotes anti-inflammatory effects, reducing the release of pro-inflammatory cytokines and regulating the release of anti-inflammatory cytokines (Boneberg and Hartung 2002). Similarly to the others CSFs, G-CSF is present in small quantities in the organism (Cebon et al. 1994), being produced by bone marrow cells, fibroblasts, endothelial cells, monocytes and macrophages (Welte et al. 1996). To facilitate the study of this cytokine, and also aiming to use this protein as a drug, G-CSF was cloned and expressed as a recombinant protein (Clark and Kamen 1987; Souza et al. 1986; Nagata et al. 1986). Nowadays, recombinant hG-CSF (rhG-CSF) is a well established commercially available biotechnological product, and one of the most widely used recombinant protein for clinical purposes. Its use is centered on the treatment of neutropenias, a state of low number of neutrophils, which may be acquired or congenital, and is mainly related to bone marrow transplantation, chemotherapy, radiotherapy, myelodysplasia, and infections, such as by HIV (Welte et al. 1996; Hubel and Engert 2003). Besides, rhG-CSF is employed to mobilize peripheral blood stem cells, allowing them to be safely harvested and used for transplantation, and also in the treatment and prevention of infections in non-neutropenic patients (Hubel and Engert 2003). There are four principal forms of G-CSF available for clinical use at the present time (Molineux 2011): Filgrastim, a recombinant G-CSF produced in Escherichia coli, which is identical to the human protein, except for being non-glycosylated and for having an additional N-terminal methionine on its amino acid composition; Lenograstim, the recombinant version of Filgrastim expressed in a mammalian expression system (CHO cells), which is, therefore, glycosylated and devoid of the extra N-terminal methionine; Nartograstim, a mutant variation of the original human protein, which is also expressed in E. coli (being, thus, non-glycosylated and having the additional N-terminal methionine), having four amino acid changes in its N-terminus and a substitution of one cysteine (Cys 17, which is not involved in the formation of disulfide bond in the active protein); and Pegfilgrastim, the only second generation G-CSF available for clinical use, which has a covalent 20 kDa polyethylene glycol moiety covalently bound to the N-terminal methionine of the Filgrastim molecule, aiming to reduce the proteolytic degradation and to avoid the renal elimination of the molecule. Although these proteins were claimed to have diverse properties, different biological activities and also differences on its pharmacological properties, this is still a matter of discussion (Tanaka et al. 1997). Currently, the patents of the first generation recombinant G-CSF proteins have expired. Filgrastim patent expired in 2006 and several rhG-CSF biosimilars are currently available in Europe, China and Asia, while some others have

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entered the European and North-American regulatory framework for approval (Molineux 2011). On the other hand, although the North-American Nartograstim patent has expired in 2010, to our knowledge, none Nartograstim biosimilar is currently available. The large clinical importance and the widespread demand for G-CSF drugs in healthcare justify efforts to achieve new functional and viable biosimilars. In Brazil, G-CSF is freely distributed by the national healthcare system (the Unified Health System, a part of the Ministry of Health) and the production of a locally developed biosimilar would thus impact on both public healthcare system (enhancing the ability/coverage/capacity of drug distribution) and governmental spendings (reducing the costs of acquiring this drug). Here, we describe the production of a Nartograstim biosimilar using a codon-optimized gene. Higher yields (15 mg/l) of the recombinant protein were obtained when compared to previously described protocols (Vanz et al. 2008; Bishop et al. 2001) and the protein showed to be as active as the commercially available rhG-CSF.

Materials and methods Gene synthesis The cDNA encoding hG-CSF (accession number E07164) was codon-optimized for expression in E. coli (Supplementary Fig. 1), based on the codon usage data of this bacterial species, available at the Kazusa database (Nakamura et al. 2000). Basically, the most frequent E. coli codons for each amino acid were used to substitute the human codons, that would be restrictive for expression in these bacteria, taking care to avoid nucleotide sequences that may form long hairpins and secondary structures. In addition, five codons were also changed (Thr-1 for Ala, Leu-3 for Thr, Gly-4 for Tyr, Pro-5 for Arg e Cys-17 for Ser), to produce the rhG-CSF version known as Nartograstim or KW-2228, a protein with a potentially greater biological activity (Kuga et al. 1989) and stability (Okabe et al. 1990). The codon-optimized gene with these five mutations was named opti-narto. Bacterial strains, plasmids and medium The E. coli strain DH5a (Life Technologies, Inc., Rockville, MD, USA) was used for all routine cloning experiments, whereas the E. coli strain BL21 (DE3) Star pLysS (Novagen) was used as a host for recombinant protein expression. The pUC57 vector containing the opti-narto synthetic gene was purchased from GenScript Corporation (Piscataway, NJ, USA), the pGEM-T easy plasmid was purchased from Promega and the pAE plasmid was developed in our laboratory


(Ramos et al. 2004). Luria–Bertani medium (LB) with 100 lg/ml ampicillin or with 100 lg/ml ampicillin and 50 lg/ml chloramphenicol was used for bacterial culture. Gene cloning All DNA manipulations were carried out as described (Sambrook et al. 1989). The DNA fragment coding for Nartograstim (opti-narto) was amplified by a polymerase chain reaction (PCR) from pUC57-opti-narto using a forward primer containing an NdeI restriction site and a reverse primer containing an EcoRI restriction site. PCR was carried out with 50 ll of reaction mixture containing 10 ng template DNA, 0.2 mM each dNTP, 20 pmol each primer, 19 PCR buffer (Invitrogen), and 0.5 U PlatinumÒ Taq DNA polymerase High Fidelity (Invitrogen). The PCR assay conditions were: 95 °C for 4 min, followed by 30 cycles of 95 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min, and then a final extension at 72 °C for 5 min. The amplified product was fractionated by electrophoresis on 1 % agarose gel, stained with ethidium bromide and visualized in a UV-transilluminator. The region containing the 550 bp band of interest was cut out from the agarose gel, and the PCR product was purified using a commercial extraction system (GFXTM PCR and Gel Band Purification, GE Healthcare). The recovered DNA was then cloned into the pGEM-T easy plasmid, following manufacturer’s instructions. Next, the DNA insert containing the optinarto gene was isolated from pGEM-T-opti-narto construction by double digestion with NdeI and EcoRI, and further cloned into the expression vector pAE, at the same restriction sites. The resulting plasmid was named pAEopti-narto, and the construction was confirmed by DNA sequencing in an ABI PRISM 3100 Genetic Analyzer (Applied Biossystems) using the ABI Prism Big Dye Terminator kit and a T7 promoter primer. Expression and purification of recombinant optiNartograstim Escherichia coli BL21 (DE3) Star pLysS cells transformed with pAE-opti-narto were grown at 37 °C in one liter of LB medium containing 100 lg/ml ampicillin and 50 lg/ml chloramphenicol. When absorbance at 600 nm reached 0.6, isopropyl-b-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM, and the cells were harvested by centrifugation 4 h later. The bacterial pellet was resuspended in 0.02 M phosphate buffer, pH 8.0, lysed with a French Press (Thermo Spectronic) at 30,000 psi, and the soluble and insoluble fractions were isolated by centrifugation at 8,400g for 15 min. The insoluble fraction, containing the inclusion bodies, was washed with 50 ml of a low chaotropic buffer (2 M urea, 0.02 M phosphate

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buffer, pH 4.5, 0.05 M NaCl) and further solubilized with 150 ml of a chaotropic buffer (8 M urea, 0.02 M phosphate buffer, pH 4.5, 0.05 M NaCl, 5 mM b-mercaptoethanol) during 24 h at room temperature with stirring, and then clarified by centrifugation (8,400g for 15 min). The clarified supernatant was subjected to a refolding process using a fivestep gradient of decreasing urea concentrations. Each dialysis step was performed for 12 h in 2 l of solution, at 4 °C. The solutions used in each step were: 4, 2, 1, 0.5 and 0 M urea, in 0.02 M phosphate buffer, pH 4.5, 0.05 M NaCl. After centrifugation (8,400g for 15 min), the supernatant obtained after the last dialysis was applied to a 5 ml SP Sepharose Fast Flow (GE Healthcare) cation exchange column. The resin was washed with 100 ml of 0.02 M phosphate buffer, pH 4.5, 0.05 M NaCl, and the recombinant protein was eluted with 0.02 M phosphate buffer, pH 8.0, 0.1 M NaCl. Although the amino acid sequence of our recombinant protein is identical to the amino acid sequence of Nartograstim, we named our recombinant protein optiNartograstim to highlight the codon-optimized gene. Production of opti-Nartograstim antiserum The antiserum was produced in six BALB/c mice by intraperitoneal injection of 10 lg of the opti-Nartograstim and 100 lg of aluminum ion (added in the form of Al(OH)3) weekly, for 4 weeks. Mice were bled in the 5th week; the blood samples were pooled and allowed to coagulate for 30 min at 37 °C. After 60 min at 4 °C, the serum was collected after a 10 min centrifugation (400g, at 4 °C). Antibody titer was determined by ELISA, using 1 lg of the opti-Nartograstim as the coating antigen. Absorbance was read at 492 nm, and the serum dilution with a reading of 0.1 was considered the serum titer. Immunoblotting Recombinant proteins or E. coli extracts were fractionated on a 12 % SDS-PAGE and electro-transferred to a nitrocellulose membrane. The membrane was blocked with 10 % (w/v) non-fat dried milk in phosphate buffered saline containing 0.05 % Tween 20 (PBS-T) at 4 °C, overnight. After three washes of 10 min with PBS-T, the membrane was incubated for 90 min with mouse anti-opti-Nartograstim serum in 5 % non-fat dried milk-PBS-T (1:1,000), at room temperature with shaking. A control experiment was done using a commercial anti-G-CSF (Peprotech). After three washes of 10 min with PBS-T, the membrane was further incubated for 60 min with goat anti-mouse IgGperoxidase conjugate (Sigma) in 5 % non-fat dried milkPBS-T at room temperature with shaking. After three more washes of 10 min with PBS-T, the membrane was developed with ECL Western Blotting detection reagent

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(GE HealthCare) and then exposed to Amersham Hyperfilm ECL (GE HealthCare). The N-terminal fragment of Pneumococcal Surface Protein C (PspC) was used as a nonrelated control protein and was a generous gift from Dr. M. L. S. Oliveira (Ferreira et al. 2009).

Circular dichroism


expressed a recombinant protein with approximately 19 kDa. Similarly to other reports on rhG-CSF expression in E. coli (Rao et al. 2008; Jevsevar et al. 2005), optiNartograstim was expressed as inclusion bodies (Fig. 1). The insoluble fraction was separated from the cell lysate by centrifugation, washed and solubilized in 8 M urea. A stepwise refolding process yielded soluble opti-Nartograstim (Fig. 2), with an overall small loss of recombinant protein due to precipitation (data not shown).

The secondary structure composition of opti-Nartograstim was assessed by circular dichroism (CD) spectroscopy, using a JASCO J-810 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan). Proteins were previously dialyzed in 20 mM sodium phosphate buffer, pH 7.4, and the measurements were performed from 190 to 260 nm, at intervals of 0.1 nm in a 0.1 cm path length cell. The CD spectrum presented is the average of five measurements, performed at 20 °C. The secondary structure content was estimated using the DichroWeb server (Whitmore and Wallace 2008) employing the CDSSTR algorithm (Compton and Johnson 1986).

In vivo biological activity assay The biological activity of G-CSF was evaluated in a cyclophosphamide-induced neutropenia murine model using C57BL/6 female mice. The neutropenia was induced in three groups of eight animals with an injection of 4.5 lg of cyclophosphamide (diluted in 100 ll saline solution). For the next 5 days, the two experimental groups received daily: 1.5 lg of opti-Nartograstim or 1.5 lg of Filgrastim (GranulokineÒ, Roche), both diluted in 100 ll saline solution; the control group received 100 ll saline solution during the same period. In an additional control group of eight animals, the neutropenia was not induced, and the mice were injected daily with 100 ll saline solution during 6 days. After the injection period, a blood sample was collected by cardiac puncture from each animal. Samples were smeared and stained with Rosenfeld stain, and the number of neutrophils, was counted directly under a microscope. Statistical significance was calculated using a one-way ANOVA followed by Mann–Whitney U test.

Fig. 1 Expression of opti-Nartograstim in the insoluble fraction. Cell lysate before and after induction, and the supernatant and insoluble fraction of the induced culture are shown in a SDS-PAGE (15 %), after staining with coomassie blue. Lanes: 1 control culture (transformed with pAE); 2 non-induced culture; 3 induced culture; 4 supernatant after centrifugation process; 5 insoluble fraction, containing opti-Nartograstim

Results Opti-Nartograstim is overexpressed as an inclusion body The expression system used in this work, based on a codonoptimized synthetic gene, led to a high expression of optiNartograstim. All clones transformed with pAE-opti-narto

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Fig. 2 Refolded opti-Nartograstim. The culture lysates, the supernatant after centrifugation and the insoluble fraction after the refolding process are shown in a SDS-PAGE (15 %) after staining with coomassie blue. Lanes: 1 control culture (transformed with pAE); 2 non-induced culture; 3 induced culture; 4 supernatant after centrifugation process; 5 insoluble fraction after the refolding process


Fig. 3 Purification of opti-Nartograstim after the refolding process. The fractions from the cation exchange purification step are shown in a SDS-PAGE (15 %) after staining with coomassie blue. Lanes: 1 loaded sample; 2 flow through; 3–5 column wash with pH 4.5 and 50 mM NaCl buffer; 6 elution with pH 8.0, and 100 mM NaCl buffer, containing purified opti-Nartograstim. MW molecular mass markers

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Fig. 4 Specificity and reactivity of the antiserum produced against opti-Nartograstim. SDS-PAGE and equivalent Western blotting are shown. a SDS-PAGE; b Western blot using a commercial anti-G-CSF (Peprotech); c Western blot using serum anti-opti-Nartograstim. Lanes: 1 purified recombinant Pneumococcal Surface Protein C (PspC); 2 purified opti-Nartograstim

High yields of purified opti-Nartograstim were obtained The refolded sample was subjected to a single chromatographic step, using a cation exchange column (Fig. 3). Only a small amount of opti-Nartograstim did not bind to the column (Fig. 3, lane 2), and the contaminant E. coli proteins were diluted and washed away during low salt and low pH washes (Fig. 3, lanes 3–5). Opti-Nartograstim was eluted using 100 mM NaCl and pH 8.0 (Fig. 3, lane 6). No visible contaminant proteins can be observed in a sample of this fraction (in a Coomassie-Blue stained SDS-PAGE). The yield of purified soluble opti-Nartograstim was 15 mg/l of culture. Production of anti-opti-Nartograstim serum The purified protein was used to produce antiserum in BALB/c mice. High titers were obtained (data not shown), and the antiserum efficiently recognized opti-Nartograstim in Western Blotting (Fig. 4). Moreover, the antiserum showed to be specific and did not recognize a non-related recombinant protein PspC (Pneumococcal Surface Protein C). A control, commercial polyclonal anti-hG-CSF, presented similar results, also detecting opti-Nartograstim but not reacting with PspC. Opti-Nartograstim is alpha-helix structured and is biologically active After refolded and purified, the opti-Nartograstim was evaluated using CD spectroscopy. The spectrum obtained indicates that opti-Nartograstim structure is composed predominantly by alpha-helix (Fig. 5), what is in accordance with previously obtained data (Fujii et al. 1997; Nagahara et al. 1990; Lu et al. 1989). The deduced

Fig. 5 Analysis of opti-Nartograstim by circular dichroism (CD) spectroscopy. The spectrum presented is an average of five scans. The calculated percentage of each secondary structure component is also shown

secondary structure is in accordance with the crystal structure of G-CSF (Hill et al. 1993). To test the biological activity of opti-Nartograstim, a cyclophosphamide-induced neutropenia murine model was used. Neutropenic-induced mice were injected with optiNartograstim or with a commercial G-CSF (Filgrastim). Both recombinant proteins were able to significantly raise the neutrophils count after four weekly doses (Fig. 6). The effect of opti-Nartograstim and the commercial G-CSF were statistically indistinguishable.

Discussion Granulocyte-colony stimulating factor is an important drug to prevent or counteract neutropenias. This state of low

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Fig. 6 Biological activity of opti-Nartograstim in a cyclophosphamide-induced neutropenia murine model. The mean and standard deviation (bars) of the percentage of neutrophils counts are shown. The statistical significance (at P \ 0.001) in the Mann–Whitney U test is indicated by an asterisk

levels of neutrophils can be inherited or triggered by several causes, such as the use of drugs (particularly anticancer), radiotherapy and diseases (HIV as an important example). We were able to produce a functional mutated recombinant G-CSF in E. coli, with high yields. The mutant G-CSF known as Nartograstim was chosen to be used in this work, since previous results suggested that this protein is more active and more thermodynamically and biologically stable than the non-mutated protein (Kuga et al. 1989; Okabe et al. 1990). Although our protein has identical amino acid sequence to Nartograstim, it was named opti-Nartograstim, to highlight the codon-optimization that was employed here. Opti-Nartograstim was obtained at 15 mg of purified protein per liter of cell culture, a yield more than 4 times higher than previous results (expressing filgrastim, the nonmutated protein) (Vanz et al. 2008; Bishop et al. 2001). This high yield may be related to three factors: high expression, efficient refolding and efficient purification. The high expression may also be associated to the use of a codon-optimized gene. The substitution of rare codons in the codon-optimized genes can significantly enhance protein expression (Fredrick and Ibba 2010; Jana and Deb 2005; Makrides 1996). Indeed, this strategy was already successfully used to produce rhG-CSF, leading to higher expression levels (Devlin et al. 1988; Jevsevar et al. 2005; Kang et al. 1995) which would be probably not possible without codon optimization, since human G-CSF cDNA contains some rare codons for E. coli (mainly the leucines at the position 152 and 168—data not shown).

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Its expression as inclusion bodies may also have contributed to the overall yield, since insoluble proteins are protected from proteases (Makrides 1996). Similarly, the potentially increased resistance of the mutated protein to proteases (Okabe et al. 1990) may also have contributed to the overall yield, because nascent proteins may be protected from proteolysis. The refolding process was optimized to enhance the overall recovery of soluble protein, leading to an overall small loss of recombinant protein due to precipitation (data not shown). It is important to notice that Cys17 was changed to Ser in opti-Nartograstim and this may have helped to increase its solubility and refolding features. In contrast to other works (Yamasaki et al. 1998; Wingfield et al. 1988; Rao et al. 2008), a single purification step was sufficient to obtain a high purified protein. The fact that almost the entire refolded sample was composed by opti-Nartograstim (Fig. 3, lane 1) may have contributed to this result. Besides, the high expression level of the recombinant protein (Fig. 1, lane 5) which led to a very high proportion of opti-Nartograstim in the insoluble fraction is also a factor to be considered. Thus, starting from a very concentrated and pure initial sample, we showed that a single purification step was sufficient to achieve a high purity level, avoiding protein loss during additional purification procedures. It must be noted that the inclusion bodies were also the starting material of all procedures for obtaining intracellular rhG-CSF in E. coli described to date (Jevsevar et al. 2005). Indeed, despite the requirement of a refolding protocol, some authors claim that the expression as inclusion bodies may be desirable in some circumstances (Sørensen and Mortensen 2005) as shown here. The in vivo biological activity of opti-Nartograstim was statistically indistinguishable from the reference drug Filgrastim (GranulokineÒ, Roche) (Fig. 6) used to treat neutropenias. Although Nartograstim was claimed to be about three times more potent than Filgrastim and also more stable physicochemically and biologically (Okabe et al. 1990; Suzuki et al. 1992; Kuga et al. 1989), we did not observe any significant difference between these proteins in the ability to increase the neutrophils number after 4 days of experiment, in a murine cyclophosphamide neutropenia-induced model. Indeed, the enhanced activity and improved pharmacokynetic profile of Nartograstim have been questioned previously. A pre-clinical study done in primates did not detected significant differences in both the activity and the pharmacokynetic properties when comparing Filgrastim with Nartograstim (Tanaka et al. 1997). Accordingly, it was reported that although Nartograstim tended to produce a greater expansion of neutrophils than Filgrastim, there was no statistical significance in this result (Katsumori et al. 2009). Additionally, the same


group that previously claimed that there were differences between Filgrastim and Nartograstim (Kuga et al. 1989; Okabe et al. 1990) also showed, in later works, that both proteins have similar pharmacokynetic profiles in vivo, using normal rats and monkeys, and, moreover, that these proteins did not show differences in biological activity in monkeys (Kuwaraba et al. 1991a, b). These contradictory data were hypothesized to be related to the use of researchonly-use products (instead of the use of commercially available molecules, whose activities are guaranteed by the manufacturer), which may have low activity (Tanaka et al. 1997). Besides, it was also pointed out that some discrepancy between the in vitro and in vivo stability results may be a consequence of the fact that the protease degradation in plasma (expected to be less intense for Nartograstim) is not a major metabolic pathway of rhG-CSF (Tanaka et al. 1997; Kuwabara et al. 1995; Tanaka and Tokiwa 1990). Finally, Tanaka and Kaneko (1992) suggested that some differences in pharmacokinetics datasets may be a result from the use of inappropriate assay methods to estimate G-CSF serum concentration. Additionally, a polyclonal antiserum against optiNartograstim was successfully produced in mice. This serum showed to be very sensible and specific, and may be useful for further development of opti-Nartograstim production, mainly in the control of the expression and purification of this recombinant protein. In conclusion, we were able to produce a purified recombinant mutated G-CSF. This protein, named optiNartograstim, was produced with high yields (15 mg/l), probably as the result of an optimized-codon synthetic gene, to an efficient refolding process and to the use of single purification step, avoiding undesirable protein loss. Opti-Nartograstim was as active as a commercially available Filgrastim. Associated to the high yields (the highest described so far in small scale conditions), this suggests that the production of this recombinant protein, using the system described here, may be viable and could be the initial step to produce the first Nartograstim biosimilar. Acknowledgments We thank Leonardo Setsuo Kobashi, Aline Marques Cavalher, Nidia Cassiano Pereira and Izilda Maria Ramos for their technical assistance and Dr. Maria Leonor Sarno de Oliveira for providing us PspC protein. We gratefully acknowledge Dr. Eliane Namie Miyaji for the English revision of the manuscript. This work was supported by grants from FAPESP, CNPq, and Fundaçaõ Butantan.

References Barreda DR, Hanington PC, Belosevic M (2004) Regulation of myeloid development and function by colony stimulating

2599 factors. Dev Comp Immunol 28:509–554. doi:10.1016/j.dci. 2003.09.010 Bishop B, Koay D, Sartorelli A, Regan L (2001) Reengineering granulocyte colony-stimulating factor for enhanced stability. J Biol Chem 276:33465–33470. doi:10.1074/jbc.M104494200 Boneberg E, Hartung T (2002) Molecular aspects of anti-inflammatory action of G-CSF. Inflamm Res 51:119–128 Cebon J, Layton JE, Maher D, Morstyn G (1994) Endogenous haemopoietic growth factors in neutropenia and infection. Br J Haematol 86(2):265–274 Clark SC, Kamen R (1987) The human hematopoietic colonystimulating factors. Science 236:1229–1237 Compton LA, Johnson WC (1986) Analysis of protein circulardichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem 155:155–167 Devlin PE, Drummond RJ, Toy P, Mark DF, Watt KWK, Devlin JJ (1988) Alteration of amino-terminal codons of human granulocyte-colony-stimulating factor increases expression levels and allows efficient processing by methionine aminopeptidase in Escherichia coli. Gene 65:13–22 Ferreira DM, Darrieux M, Silva DA, Leite LC, Ferreira JM Jr, Ho PL, Miyaji EN, Oliveira ML (2009) Characterization of protective mucosal and systemic immune responses elicited by pneumococcal surface protein PspA and PspC nasal vaccines against a respiratory pneumococcal challenge in mice. Clin Vaccine Immunol 16:636–645. doi:10.1128/CVI.00395-08 Fredrick K, Ibba M (2010) How the sequence of a gene can tune its translation. Cell 141:227–229. doi:10.1016/j.cell.2010.03.033 Fujii I, Nagahara Y, Yamasaki M, Yokoo Y, Itoh S, Hirayama N (1997) Structure of KW-2228, a tailored human granulocyte colony-stimulating factor with enhanced biological activity and stability. FEBS Lett 410:131–135 Hill CP, Osslund TD, Eisenberg D (1993) The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc Natl Acad Sci USA 90:5167–5171 Hubel K, Engert A (2003) Clinical applications of granulocyte colony-stimulating factor: an update and summary. Ann Hematol 82:207–213. doi:10.1007/s00277-003-0628-y Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298. doi:10.1007/s00253-004-1814-0 Jevsevar S, Gaberc-Porekar V, Fonda I, Podobnik B, Grdadolnik J, Menart V (2005) Production of nonclassical inclusion bodies from which correctly folded protein can be extracted. Biotechnol Prog 21:632–639. doi:10.1021/bp0497839 Kang SH, Na KH, Park JH, Park CII, Lee SY, Lee YI (1995) High level expression and simple purification of recombinant human granulocyte colony-stimulating factor in E. coli. Biotechnol Lett 17:687–692. doi:10.1007/BF00130351 Katsumori T, Yoshino H, Hayashi M, Takahashi K, Kashiwakura I (2009) Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from X-irradiated human peripheral blood hematopoietic progenitor cells. Biol Pharm Bull 32:1849–1853. doi:10.1248/bpb.32.1849 Kuga T, Komatsu Y, Yamasaki M, Sekine S, Miyaji H, Nishi T, Sato M, Yokoo Y, Asano M, Okabe M (1989) Mutagenesis of human granulocyte colony stimulating factor. Biochem Biophys Res Commun 159:103–111 Kuwabara T, Ishikawa Y, Kobayashi H, Kobayashi S, Sugiyama Y (1995) Renal clearance of a recombinant granulocyte colonystimulating factor, nartograstim, in rats. Pharm Res 12: 1466–1469 Kuwaraba T, Kato Y, Okumura S, Kobayashi S, Yamamoto M, Ikenaga T, Deguchi T, Hirata T (1991a) Disposition of Marograstim (KW2228) (4): pharmacokinetic studies in monkeys. Drug Metab Pharmacokinet 6:887–897. doi:10.2133/dmpk.6.887

123

2600 Kuwaraba T, Okumura S, Kobayashi S, Hirata T (1991b) Disposition of Marograstim (KW-2228) (3): pharmacokinetic studies in rats. Drug Metab Pharmacokinet 6:875–885. doi:10.2133/dmpk.6.875 Li YM, Karlin A, Loike JD, Silverstein SC (2002) A critical concentration of neutrophils is required for effective bacterial killing in suspension. Proc Natl Acad Sci USA 99:8289–8294. doi:10.1073/pnas.122244799 Lu HS, Boone TC, Souza LM, Lai PH (1989) Disulfide and secondary structure of recombinant human granulocyte colony-stimulating factor. Arch Biochem Biophys 268:81–92 Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538 Molineux G (2011) Granulocyte colony-stimulating factors. In: Lyman GH, Dale DC (eds) Hematopoietic growth factors in oncology. Springer, New York, pp 33–53 Nagahara Y, Konishi N, Yokoo Y, Hirayama N (1990) Crystallization and preliminary diffraction studies of recombinant human granulocyte-stimulating factor (KW2228). J Mol Biol 214: 25–26. doi:10.1016/0022-2836(90)90143-A Nagata S, Tsuchiya M, Asano S, Kaziro Y, Yamazaki T, Yamamoto O, Hirata Y, Kubota N, Oheda M, Nomura H (1986) Molecular cloning and expression of cDNA for human granulocyte colonystimulating factor. Nature 319:415–418. doi:10.1038/319415a0 Nakamura Y, Gojobori T, Ikemura T (2000) Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucleic Acids Res 28:292. doi:10.1093/nar/28.1.292 Okabe M, Asano M, Kuga T, Komatsu Y, Yamasaki M, Yokoo Y, Itoh S, Morimoto M, Oka T (1990) In vitro and in vivo hematopoietic effect of mutant human granulocyte colonystimulating factor. Blood 75(9):1788–1793 Ramos CRR, Abreu PAE, Nascimento ALTO, Ho PL (2004) A highcopy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide. Braz J Med Biol Res 37:1103–1109. doi:10.1590/ S0100-879X2004000800001 Rao D, Narasu M, Rao A (2008) A purification method for improving the process yield and quality of recombinant human granulocyte colony-stimulating factor expressed in Escherichia coli and its characterization. Biotechnol Appl Biochem 50:77–87. doi: 10.1042/BA20070130 Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Sica S, Rutella S, Di Mario A, Salutari P, Rumi C, Ortu la Barbera E, Etuk B, Menichella G, D’Onofrio G, Leone G (1996) rhG-CSF in healthy donors: mobilization of peripheral hemopoietic progenitors and effect on peripheral blood leukocytes. J Hematother 5:391–397

123

World J Microbiol Biotechnol (2012) 28:2593–2600 Sørensen HP, Mortensen KK (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 4:1. doi:10.1186/1475-2859-4-1 Souza LM, Boone TC, Gabrilove J, Lai PH, Zsebo KM, Murdock DC, Chazin VR, Bruszewski J, Lu H, Chen KK (1986) Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 232:61–65 Suzuki T, Tohda S, Nagata K, Imai Y, Murohashi I, Nara N (1992) Enhanced effect of mutant granulocyte-colony-stimulating factor (KW-2228) on the growth of normal and leukemic hemopoietic progenitor cells in comparison with recombinant human granulocyte-colony-stimulating factor (G-CSF). Acta Haematol 87:181–189 Tanaka H, Kaneko T (1992) Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in mice. Blood 79: 536–539 Tanaka H, Tokiwa T (1990) Influence of renal and hepatic failure on the pharmacokinetics of recombinant human granulocyte colonystimulating factor (KRN8601) in the rat. Cancer Res 50: 6615–6619 Tanaka H, Tanaka Y, Shinagawa K, Yamagishi Y, Ohtaki K, Asano K (1997) Three types of recombinant human granulocyte colonystimulating factor have equivalent biological activities in monkeys. Cytokine 9:360–369. doi:10.1006/cyto.1996.0177 Vanz ALS, Renard G, Palma MS, Chies JM, Dalmora SL, Basso LA, Santos DS (2008) Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization. Microb Cell Fact 7:13. doi:10.1186/1475-2859-7-13 Weisbart RH, Golde DW (1989) Physiology of granulocyte and macrophage colony-stimulating factors in host defense. Hematol Oncol Clin North Am 3:401–409 Welte K, Gabrilove J, Bronchud M, Platzer E, Morstyn G (1996) Filgrastim (r-metHuG-CSF): the first 10 years. Blood 88:1907– 1929 Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89:392–400. doi:10.1002/bip.20853 Wingfield P, Benedict R, Turcatti G, Allet B, Mermod JJ, DeLamarter J, Simona MG, Rose K (1988) Characterization of recombinantderived granulocyte-colony stimulating factor (G-CSF). Biochem J 256:213–218 Yamasaki M, Konishi N, Yamaguchi K, Itoh S, Yokoo Y (1998) Purification and characterization of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivatives: KW-2228 and other derivatives. Biosci Biotechnol Biochem 62: 1528–1534