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Journal of Biotechnology 145 (2010) 334–340

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Synthesis, purification and characterization of recombinant glycosylated humanprolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

S.R. Heller, H. Rodrigues Goulart, F.S. Arthuso, T.L. Oliveira, P. Bartolini, C.R.J. Soares ∗

Biotechnology Department, IPEN-CNEN, Av. Lineu Prestes, 2242, Cidade Universitária, 05508-900 São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 15 May 2009Received in revised form 6 October 2009Accepted 31 December 2009

Keywords:Human prolactinGlycosylated human prolactinhPRLG-PRLCycloheximideCHO cells

a b s t r a c t

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activitieswhich is best known for its stimulation of lactation and development of mammary gland. This proteincontains only one potential asparagine-linked glycosylation site, which is partially (10–30%) occupiedwhen the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL(G-hPRL) has been found to be ∼4-fold lower than that of hPRL, its physiological function is not yet welldefined. In order to better characterize and study this hormone variant, we carried out its laboratory scalepurification from conditioned medium of genetically modified CHO cells that had been supplementedwith cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL ∼4-foldand the glycosylated versus non-glycosylated hPRL concentration ratio by ∼7-fold. G-hPRL purificationwas carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC)column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioas-say based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11–15 IU mg−1) inthe two assays. Our results show that addition of cycloheximide can be an important strategy for increas-ing glycosylated protein production, facilitating the purification and characterization of these isoforms.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Glycosylated human prolactin (G-hPRL) was first isolated andpurified from human pituitaries by Lewis et al. (1985), with anestimated molecular mass of ∼25,000 Da and an immunologicaland biological activity of 25–50% that of non-glycosylated (NG)-hPRL (Pellegrini et al., 1988; Lewis et al., 1989; Sinha et al., 1991;Hoffmann et al., 1992; Price et al., 1995; Soares et al., 2000).The presence of a unique and partially occupied glycosylationsite in Asn-31 in human, monkey, ovine, porcine, dromedary,equine and whale PRL (Sinha, 1995; Butnev et al., 1996) makesit an ideal model of glycosylation for N-glycan studies since itexhibits the simplest type of glycosylation macroheterogeneity,with an occupancy range of 10–30% of G-hPRL relative to thetotal hPRL of either pituitary or recombinant origin (Shelikoffet al., 1994, 1996; Price et al., 1995; Sinha, 1995; Soares et al.,

Abbreviations: CHO, Chinese hamster ovary; CRS, chemical reference standard;hPRL, human prolactin; HPSEC, size-exclusion HPLC; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Mr, relativemolecular mass; PAGE, polyacrylamide-gel electrophoresis; SDS, sodium dodecylsulfate; RP-HPLC, reversed-phase HPLC; tR, retention time.

∗ Corresponding author. Tel.: +55 11 31339699; fax: +55 11 31339694.E-mail address: crsoares@ipen.br (C.R.J. Soares).

2000). Unfortunately, the physiological significance of this iso-form has not yet been well elucidated, even though it has beenpostulated that hPRL glycosylation might possibly modulate thebioactivity of the circulating pool of the hormone, perhaps byselectively down-regulating PRL action at individual target tis-sues (Sinha, 1995; Freeman et al., 2000; Goffin et al., 2002). Forthese reasons, we and others believe that this and other pro-lactin variants are needed in purified and characterized form inorder to develop proper physiological and clinical studies directedtowards the understanding of the more than 300 separate actionsof this hormone, whose receptor distribution has been defined asbeing “quasi-ubiquitous” (Young et al., 1990; Price et al., 1995;Sinha, 1995; Freeman et al., 2000; Soares et al., 2000; Goffin et al.,2002).

Impetus for the present investigation was provided by the workof Shelikoff et al. (1994), who demonstrated that the addition ofa protein synthesis inhibitor (cycloheximide) to C127 cell cultureincreased recombinant hPRL glycosylation site occupancy. Our goalwas to test the effect of cycloheximide on genetically modifiedCHO cells and then purify the secreted recombinant hPRL via twochromatographic steps. Purified G-hPRL was then characterized byRP-HPLC and HPSEC, Western blotting, MALDI-TOF-MS and twoin vitro bioassays. The first is the classical heterologous Nb2-ratlymphoma-cell-proliferation assay, while the second is the homol-ogous Ba/F3-LLP assay, based on murine pro-B cells containing the

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sequence encoding the human PRL receptor (Tanaka et al., 1980;Soares et al., 2006).

2. Material and methods

2.1. Cell cultivation

The clone expressing hPRL, derived from CHO dhfr- cells (DUKX-B11) that had been transfected with the vector pEDdc-hPRL, wasobtained in our Laboratory (Soares et al., 2000). Cells were thawedinto T-flasks (75 cm2, from Corning Costar Corporation, Cambridge,MA) containing �-MEM with l-glutamine and without ribonucleo-sides and deoxyribonucleosides, supplemented with 10% dialyzedfetal bovine serum (dFBS), 40 �g ml−1 gentamycin, 4 g l−1 glucoseand 100 nM methotrexate. Cultures were maintained in a humidi-fied incubator at 37 ◦C in the presence of 5% CO2. After two passages,cells were seeded (1–2 × 105 cells ml−1) into six-well-dishes orT-flasks used in the experiments and for laboratory production.When cells were confluent, cultures were rinsed twice in phosphatebuffered saline to remove serum and the same medium, but with-out serum, methotrexate and with cycloheximide (0–2 �g ml−1),was added to the culture. G-hPRL-secreting confluent cultures weremaintained up to three weeks by replacing the medium every 24 h.Collected conditioned medium was stored at −80◦C.

2.2. G-hPRL purification

The G-hPRL present in conditioned culture medium was puri-fied using a method described by Soares et al. (2006). A two-steppurification process was used: SP-Sepharose Fast Flow followed bya size-exclusion chromatography employing HPSEC for prepara-tive purposes. Briefly, conditioned medium (∼500 ml) was adjustedto pH 5.0 using acetic acid. The material was then applied ontothe SP-Sepharose Fast Flow column (Pharmacia, São Paulo, Brazil)equilibrated in 50 mM sodium acetate (pH 5.0). UV absorbancewas monitored at 280 nm. After washing with the same buffer,the column was washed with 50 mM sodium acetate (pH 5.0),90 mM NaCl. The protein of interest was then eluted from the col-umn with 25 mM Hepes (pH 8.0). Different fractions containinghPRL were analyzed by HPSEC and the most concentrated frac-tions purified on the same HPSEC column, working this time as apreparative column. The maximum volume applied for each HPSECpreparative run was 500 �l. A final pool, derived from HPSEC purifi-cation, was quantified for bioassay purposes via HPSEC and storedat–80 ◦C.

2.3. Gel electrophoresis and Western-blot analysis

Discontinuous SDS-PAGE, based on 15% polyacrylamide gels,was carried out under non-reducing conditions as described byLaemmli (1970). Coomassie Brilliant Blue G-250 (USB, Cleveland,OH) was used for staining and the molecular mass markers werefrom GE Healthcare BioSciences (Uppsala, Sweden). Western blotanalysis was performed according to Bannerman et al. (2002), usingProtein A, radioiodinated in our laboratory as described by Ribela etal. (1996). Gels were loaded with equal proportions of conditionedmedia.

2.4. RP-HPLC

A Shimadzu Model SCL-10A HPLC apparatus coupled to a SPD-10AV UV detector (Shimadzu, MD, USA) was used, employing theClass VP software, also from Shimadzu. The column was a C4 Vydac214TP54 (25 cm × 4.6 mm ID, pore diameter of 300 Å and particlediameter of 5 �m) with a silica precolumn packed with LiChrosorbSi-60, 7.9–12.4 �m (Merck, Darmstadt, Germany) located between

Fig. 1. Western blot analysis showing the effect of cycloheximide on hPRL glycosy-lation. Confluent CHO cell cultures were harvested daily, the 3rd day fraction beinganalyzed. Lane 1, hPRL internal reference preparation from E. coli; lane 2, regularcultivation without cycloheximide; lanes 3–7, cultures with 0.02, 0.06, 0.2, 0.6 and2 �g ml−1 cycloheximide addition, respectively.

the pump and the injector. Vydac columns were purchased fromGrace Vydac (Hesperia, CA, USA). The mobile phase consisted of 71%Tris–HCl buffer (50 mM, pH 7.5) and 29% n-propanol, as describedby Dalmora et al. (1997), with a flow-rate of 0.5 ml min−1, detectorwavelength at 220 nm, column temperature maintained at 45◦Cand a sample volume of 25–200 �l (Soares et al., 2002).

2.5. HPSEC

HPSEC was carried out with the same Shimadzu apparatus,processing 5–500 �l of sample on a TosoHaas (Montgomeryville,

Fig. 2. Western blot analysis of hPRL secretion from CHO cells during a 6-day pro-duction in culture flasks, after addition of 0.6 �g ml−1 cycloheximide to the medium.IRP, internal reference preparation of hPRL from E. coli. Lanes 1–6, conditioned mediafrom day 1 to day 6, respectively.

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Fig. 3. SP-Sepharose FF purification of hPRL from conditioned medium of hPRL-secreting CHO cells. The hPRL peak was confirmed by Western blotting.

Fig. 4. HPSEC analysis of partially purified hPRL obtained after SP-Sepharose FFchromatography. (A) Product obtained after culturing CHO cells in the presenceof 0.6 �g ml−1 cycloheximide. (B) Control product obtained without cycloheximideaddition. (C) Chemical Reference Standard (CRS) of hPRL from WHO.

Fig. 5. HPSEC purification on a Tosohaas G2000 SW column (60 cm × 7.5 mm i.d.) ofthe peak fraction obtained from SP-Sepharose FF (A) and Western blot analysis of thecorresponding fractions eluted from HPSEC (B). IRP, internal reference preparationof hPRL from E. coli.

PA, USA) G2000 SW column (60 cm × 7.5 mm i.d., particle size of10 �m and pore size of 125 Å) coupled to a 7.5 cm × 7.5 mm i.d. SWguard column. The mobile phase was 0.025 M ammonium bicar-bonate, pH 7.0, with a flow-rate of 1.0 ml min−1 (Dalmora et al.,1997). This methodology was used for analytical and preparativepurposes.

2.6. In vitro bioassays

The biological activities of purified G-hPRL were determinedusing the Nb2 and BaF3-LLP lymphoma-cell-proliferation assays,as previously described (Glezer et al., 2006), against the First WHOReference Reagents of recombinant hPRL and recombinant G-hPRL,lyophilized in ampoules coded 97/714 and 98/580, respectively(Rafferty et al., 2001). Sample quantification was carried out viaHPSEC against the WHO Chemical Reference Standard (CRS) ofhPRL.

2.7. Mass spectrometry

MALDI-TOF-MS analyses of hPRL (NG-hPRL/G-hPRL and G-hPRL)were carried out by Commonwealth Biotechnologies, (Richmond,VA, USA) using sinapinic acid (SA) as the matrix and a Voyager-DE BioSpectrometry Work-station from Applied Biosystems (FosterCity, CA, USA).

2.8. Glycosidase digestion of G-hPRL

Purified G-hPRL, obtained by the method described above, wassubjected to peptide N-glycosidade F treatment to confirm the pres-ence of sugar chains. A sample of 1.25 �g of purified G-hPRL wasboiled for 10 min in 10 �l of denaturing buffer (0.5% SDS, 0.04 MDTT). Following addition of 500 units of peptide N-glycosidase F(Biolabs New England) and reaction buffer (0.05 M sodium phos-phate and 1% Noninet P-40), the mixture (20 �l) was incubated 4 hat 37 ◦C and then subjected to Western Blotting.

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Fig. 6. HPSEC analysis of final purified G-hPRL (A) and of the Chemical Reference Standard of hPRL (WHO-CRS) (B). (C) Western blot analysis of G-hPRL from differentpurification steps. 1, internal reference preparation of hPRL from E. coli; 2, CHO conditioned medium; 3, fraction eluted from SP-Sepharose FF; 4, purified G-hPRL eluted fromHPSEC; 5, 1st Reference Reagent for G-hPRL (WHO 98/580); 6, WHO-CRS, showing the presence of G-hPRL and NG-hPRL. (D) N-glycosydase F digestion of G-hPRL. 1, hPRLfrom E. coli; 2, WHO-CRS; 3, glycosydase-treated G-hPRL; 4, untreated G-hPRL.

3. Results

The concentration of cycloheximide to be added to the culturemedium for obtaining the highest proportion of G-hPRL was deter-mined by Western blotting in the experiment presented in Fig. 1. Aworking concentration of 0.6 �g cycloheximide ml−1 (lane 6) waschosen, since at higher concentrations some morphological alter-ations of the cells, with consequent loss of viability, were observed.This condition was also used for the 6-day production cycle illus-trated in Fig. 2. We note that, in this production cycle, the first twodays are not useful for G-hPRL production; consequently, harvest-ing was initiated in general on day-3. The production cycle couldthen be extended up to 15 days, while still providing a productadequately enriched in G-hPRL.

A first concentration/purification step was carried out on acationic column (SP-Sepharose FF), providing a single fraction thatcontained both NG-hPRL and G-hPRL, as confirmed by Westernblot analysis (Fig. 3). Since it is well known that Western blottingquantification is not sufficiently accurate, two CHO conditioned

media, one with and one without cycloheximide addition, werepartially purified in parallel by SP-Sepharose FF and then ana-lyzed qualitatively and quantitatively by HPSEC and RP-HPLC. Anexample of this comparison is shown in Fig. 4, which also includesthe chromatogram of the Chemical Reference Standard from WHOthat contains the two hPRL isoforms (Rafferty et al., 2001). Thisprovided acceptable accuracy and the presence of cycloheximidein n = 3 experiments increased secretion of G-hPRL in the con-ditioned medium by ∼4-fold, from a total of 25.4 ± 4.8 �g l−1 to113 ± 54 �g l−1 (Fig. 4) or in mass fraction from 0.16 ± 0.07 to0.57 ± 0.18 on total prolactin. The G-hPRL to NG-hPRL concen-tration ratio thus went from 0.19 to 1.32, corresponding to a∼7-fold increase. At the same time cycloheximide greatly reducedthe presence of contaminant proteins, possibly by protein syn-thesis inhibition in general, particularly visible after SP-Sepharosepurification (Fig. 4). This is greatly facilitating G-hPRL purification.Volumetric contents in terms of total prolactin (NG-hPRL + G-hPRL)are in general of the order of 200–1000 �g l−1 of conditionedmedium. Overall recovery yields of either NG-hPRL or G-hPRL were

Fig. 7. Example of in vitro bioassays based on Ba/F3-LLP (A) and Nb2 (B) cells. (�) 1st WHO Reference Reagent for hPRL 97/714 (57.2 IU mg−1); (*) 1st WHO Reference Reagentfor G-hPRL 98/580 (16 IU mg−1) and (�) G-hPRL-IPEN.

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Fig. 8. MALDI-TOF-MS analysis of the partially purified fraction eluted from SP-Sepharose FF and containing G-hPRL and NG-hPRL (A), of purified G-hPRL (B) and of theChemical Reference Standard - WHO containing G-hPRL and NG-hPRL (C).

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of the order of 30–60%, the lowest yields being obtained in the caseof G-hPRL from untreated cells, due to the low mass fraction beingpresent in this case.

A final HPSEC purification of the fraction obtained from SP-Sepharose FF is shown in Fig. 5, where the eluted fractions wereanalyzed by Western blotting; the results confirm that a usefulseparation between the two isoforms is obtained. The chromato-graphic purity of G-hPRL was confirmed by analytical HPSEC(Fig. 6A) versus the Chemical Reference Standard (Fig. 6B). The peakwith a tR = 21.50 min on HPSEC is due to the buffer. ComparativeWestern blots of the different purification steps and of the refer-ence preparations are also shown in Fig. 6C. In Fig. 6D the identityof G-hPRL is confirmed by partial digestion with N-glycosydase F,illustrated in comparison with undigested G-hPRL and two refer-ence preparations.

The biological activities of purified G-hPRL, determined via invitro bioassays with Nb2 and Ba/F3 cells, were 14.7 ± 5.70 IU mg−1

and 11.2 ± 1.54 IU mg−1, respectively (Fig. 7). The potency deter-mined via the Nb2 bioassay was not significantly different (P > 0.05)from the value of 16.0 ± 5.25 (n = 5, CV = 32.8%) determined by a“WHO International Collaborative Study on the glycosylated andnon-glycosylated components of recombinant DNA-derived pro-lactin” (Rafferty et al., 2001).

MALDI-TOF-MS determination (Fig. 8A), carried out first on theSP-Sepharose FF eluted fraction containing the two prolactin iso-forms, showed a major peak of NG-hPRL with a relative molecularmass (Mr) of 22,888.8, i.e., different by only −0.04% from the calcu-lated theoretical value (Soares et al., 2006, 2008), as well as severalminor peaks. The heterogeneous peak with the central form pre-senting a Mr of 24,640, was apparently G-hPRL. Purified G-hPRL,analyzed in a separate experiment, provided a cluster of differ-ent isoforms, in the Mr range 24,013–25,454, the central formwith Mr 24,970 (Fig. 8B). The Chemical Reference Standard fromWHO presented, as expected, two clean peaks, although with somemicroheterogeneity, with Mr 22,880 (NG-hPRL) and 25,138 (G-hPRL) (Fig. 8C).

4. Discussion

For the first time, G-hPRL has been obtained as the predomi-nant isoform via addition of cycloheximide to a CHO cell culture.As found by Shelikoff et al. (1994) with mouse C127 cells, a con-centration of 0.6 �g ml−1 cycloheximide appeared to be an optimalconcentration of the protein synthesis inhibitor; likewise, the frac-tions of secreted G-hPRL increased in a parallel way, i.e., from 20%to 80% in their study and from 16% to 57% in the present work.In our case, however, pre-incubation with cycloheximide was notperformed, but the effect of the inhibitor was still observable upto 15 days of culture. Shelikoff et al. (1994), who studied more theglycosylation event in itself and site occupancy, tested a battery ofsix protein synthesis inhibitors, following, however, their effectsonly up to 1–2 days.

In the present study, G-hPRL obtained by cycloheximide addi-tion was purified for comparison with a control product obtainedwithout inhibitor and their absolute amounts were accuratelyquantified via HPSEC. G-hPRL secretion was found to increase ∼4-fold and, at the same time, the mass fraction of this isoform presentin the total prolactin was also greatly enriched, hence facilitatingits purification and characterization. SP-Sepharose FF was found tobe a practical concentration/purification step that allowed prelim-inary characterization of the G-hPRL/NG-hPRL mixture. The 60 cmlong HPSEC column also proved to be a flexible tool useful for boththe final purification and the qualitative and quantitative analy-sis of purified G-hPRL. It also was capable of separating the twoisoforms in a run of <30 min. In comparison with Price’s (1995)purification technique we used two instead of three purification

steps, obtaining practically the same purity in much less time. Ourscale is, however, smaller, even though we obtained similar yields.Following the same philosophy we also preferred to recover all gly-cosylated subpopulations, not selecting some particular forms ascould be done by using lectin affinity chromatography.

MALDI-TOF-MS, carried out on the heterogeneous product,allowed identification and relative molecular mass determinationsfor NG-hPRL and G-hPRL confirming previously reported values(Price et al., 1995; Soares et al., 2006). Purified G-hPRL indicatedthe presence of different molecular isoforms in approximately thesame range of those (Mr 24,864–25,588) that were determined byPrice et al. (1995) via electrospray ionization mass spectroscopyfor recombinant C127 cell-derived G-hPRL (Genzyme). It is inter-esting to note that the differences in Mr between the main G-hPRLisoforms and NG-hPRL correspond to mass values (1770, 2082 and2367) that are perfectly compatible with the masses of frequentlyfound N-glycans, such as those determined in previous work onhTSH (Oliveira et al., 2008).

As in a previous study (Glezer et al., 2006), two different in vitrobioassays were utilized to assess the biological activity of purifiedG-hPRL, confirming a potency 4–5-fold lower in comparison withNG-hPRL, as reported in the WHO International Study (Rafferty etal., 2001).

It has been reported that cycloheximide inhibits protein synthe-sis by reducing the elongation rate of ribosomes along the mRNAcoding for hPRL, thereby extending the time available for the uniquesite to become glycosylated (Shelikoff et al., 1994). Because thisaltered condition might also affect glycosylation as a whole, morestudies are needed in order to determine whether a substantialqualitative alteration occurs in the single N-linked oligosaccharideof G-hPRL. Thus, using this simple glycoprotein model, might alsobe possible to gain better insight into details of the glycosylationprocess itself.

Acknowledgments

We are grateful to João Ezequiel de Oliveira and José Maria deSousa for valuable and skilled technical assistance. This work wassupported by FAPESP, São Paulo, Brazil (Projects 05/60826-3, 06/52973-9, 07/59709-8, 07/59540-3, 07/54493-7); by the NationalResearch Council (CNPq), Brasília, Brazil (PQ 301103/2006-2) andby FK Biotecnologia/Proteogenética (RS-Brazil).

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