This article was downloaded by: [Moskow State Univ Bibliote]On: 10 December 2013, At: 04:34Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Animal BiotechnologyPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/labt20

Human Ubiquitin C Promoter BasedExpression of Erythropoietin in CHOK1 Cell Lines: A Simple TransfectantsScreening ApproachMurugan Surendarbabu a & Sankaranarayanan Meenakshisundaram aa Center for Biotechnology, Anna University, Guindy , Chennai , IndiaPublished online: 18 Jun 2013.

To cite this article: Murugan Surendarbabu & Sankaranarayanan Meenakshisundaram (2013) HumanUbiquitin C Promoter Based Expression of Erythropoietin in CHO K1 Cell Lines: A Simple TransfectantsScreening Approach, Animal Biotechnology, 24:3, 198-209, DOI: 10.1080/10495398.2013.766617

To link to this article: http://dx.doi.org/10.1080/10495398.2013.766617

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

HUMAN UBIQUITIN C PROMOTER BASEDEXPRESSION OF ERYTHROPOIETIN IN CHO K1 CELLLINES: A SIMPLE TRANSFECTANTS SCREENINGAPPROACH

Murugan Surendarbabu andSankaranarayanan MeenakshisundaramCenter for Biotechnology, Anna University, Guindy, Chennai, India

Erythropoietin (EPO), a glycoprotein hormone that regulates the production of

erythrocytes in the human body, is of clinical importance in the treatment of anemia.

Low expression levels of this recombinant hormone and time-consuming screening methods

have made its commercial production expensive. Cloning of human EPO gene in a shuttle

vector pUB6/V5-HisB driven by human ubiquitin C promoter and its transfection in CHO

K1 cell lines by electroporation resulted in a moderate level of EPO expression. The

limiting-dilution screening method required several months to obtain high expression stable

transfectants but needed only short duration for selection in contrast to the present screen-

ing strategy. The supernatants of stably transfected cells were found to be biologically

active by in vitro erythroid cluster forming activity.

Keywords: CHO K1; Erythropoietin; Transfection; Ubiquitin C promoter

Protein therapeutics is a fast growing segment in the biopharmaceutical industry,which has an annual turnover of over US $57 billion. The production of recombi-nant glycoproteins requires the use of mammalian cells such as Chinese hamsterovary (CHO) cell lines, for the purpose of protein folding and post-translationalmodification (PTM) and their biological activity (1). CHO cell lines have been usedwidely in many biomedical applications ranging from large scale production ofrecombinant human-like protein therapeutics to analyze the cellular metabolismsand toxicology studies. With regard to their wide application these cell lines aretermed as the mammalian equivalent of the model bacterium, E. coli (2). Themajority of human pathogenic viruses including HIV, influenza, polio, herpes, andmeasles do not replicate in CHO (3), and engineering of glycosylation enzymes havebeen well-documented, especially in erythropoietin (EPO), for providing enhancedproduct quality (4).

I amvery thankful toDr.C.D.Anuradha (Associate Professor) for givingme the opportunity towork in

her cell culture laboratory, CBT, AnnaUniversity, Chennai, and to S.Mubeen Fathima (Research Scholar) for

her help in working with the stem cells.

Address correspondence to Sankaranarayanan Meenakshisundaram, Center for Biotechnology,

Anna University, Guindy, Chennai 600025, India. E-mail: meenakshi@annauniv.edu

Animal Biotechnology, 24: 198–209, 2013

Copyright # Taylor & Francis Group, LLC

ISSN: 1049-5398 print=1532-2378 online

DOI: 10.1080/10495398.2013.766617

198

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

The EPO is a glycoprotein hormone that regulates the production oferythrocytes in mammals; it is one of the most important and widely used thera-peutic proteins (5). Recombinant human EPO (rh-EPO) has revolutionized thetherapeutic approaches in treating the patients with anemia in chronic renal disease.Clinical studies have demonstrated the importance of rh-EPO in various uremic con-ditions including hematological and oncological disorders, prematurity, HIV infec-tion, and other therapies (6). Expressions of rh-EPO in different vectors werecarried out in various expression systems, including Escherichia coli (E.coli) (7, 8),Pichia pastoris (P. pastoris) (9), Baculovirus (10, 11), and mammalian expression sys-tem (12–14). Attempts to use various promoters like CMV, SV40, and so forth, forthe expression of different recombinant proteins in mammalian expression systemhave been found to be effective. Like other promoters, the human ubiquitin C pro-moter has a wide range of applications and it is active in regulating the expression ofexogenous genes after transfection with appropriate expression vectors in differentcell lines (15).

Blasticidin resistance has been successfully used as a selection marker in thetransfection of mammalian cell lines. In transfection, the resistant colonies wereselected through expression of the Aspergillus blasticidin-S deaminase, which con-verts blasticidin S to a nontoxic deaminohydroxy derivative. Blasticidin-S HCl is anucleoside antibiotic, isolated from Streptomyces griseochromo genes, which inhibitprotein synthesis in both prokaryotic and eukaryotic cells (16, 17). The lowexpression levels and the limited-dilution method require several months to obtainhighly productive rh-EPO cells, which makes its commercial production expensive.The present study is an attempt to establish a stable, hyperexpression CHO K1 cellline, with less explored human ubiquitin C promoter for recombinant protein pro-duction. To achieve the over-expression of rh-EPO driven by human ubiquitin Cpromoter, it involves cloning the human EPO gene comprised of 583 bp with a27 amino acid N-terminal signal sequence, in a shuttle vector pUB6=V5-HisB, fol-lowed by stable transfection of the linearized recombinant construct (pUB6=V5-HisB-EPO) in CHO K1 cell lines. This simple screening strategy was effectiveand employed for the selection of high producer clones.

MATERIALS AND METHODS

Chemicals and Reagents

Molecular biology reagents were purchased from New England Biolabs, UK.Plasmid extraction and purification kits were purchased from Qiagen, Germany,and were used for the construction of vector and its transfection. Media componentsfrom Himedia, India, were used for culturing cell lines and fetal bovine serum (FBS)from Gibco was used for medium supplementation.

Strains and Plasmids

BHK cell line harboring the human EPO cDNA clone was obtained fromAmerican Type Culture Collection (ATCC), USA. An adherent CHO K1 cell linefrom National Center for Cell Science (NCCS), Pune, was used for the expression

HUMAN UBIQUITIN C PROMOTER BASED EXPRESSION 199

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

of recombinant human EPO. E.coli strain Top10 F’ was used for construction andpropagation of the shuttle vector. Plasmid pUB6=V5-HisB was obtained fromInvitrogen (Carlsbad, CA, USA).

Recombinant pUB6/V5-HisB-EPO Construction

All molecular biology methods were performed as described in Sambrook andRussell (18). The BHK cell line was grown in DMEM medium supplemented withFBS to maximum confluency and the genomic DNA was extracted. Gene specificprimers were used to amplify EPO gene from the genomic DNA template withPCR. The EPO gene was amplified using forward primer flanked with HindIIIand reverse primer flanked with ApaI.

EPO forward: 50-ACG AAG CTT ACG ATG GGG GTG CAC GAA TG-30

EPO reverse: 50-CGC GGG CCC TCA TCT GTC CCC TGT CCT GCA G-30

The PCR amplified EPO gene was cloned between HindIII and ApaI restrictionsites into vector pUB6V5HisB under the control of ubiquitin promoter. Genesequencing was done at MWG Biotech, India. Sequencing was performed with theprimers as follows:

UB forward priming site: 50-TCAGTGTTAGACTAGTAAATTG-30 (1167-1188)BGH reverse priming site: 50-TAGAAGGCACAGTCGAGG -30 (1418-1435)

Blasticidin Resistance for CHO K1 Cells

To generate a stable cell line expressing the target protein, it is necessary todetermine the minimum concentration of blasticidin required to kill the untrans-fected host cell line. CHO K1 cell line was grown in Hams F12 medium supplemen-ted with 10% FBS for 50% confluency and the blasticidin resistance for the cell linewas determined by applying different concentrations (from 2 to 10 mg=mL) of theantibiotic blasticidin.

Stable Transfection of CHO K1 Cell Lines

The cell electroporation was performed by a method reported earlier by Takagi(19). After cells reached 50–70% confluency, CHO K1 cells were detached using tryp-sin and resuspended in electroporation phosphate buffered saline to concentration of�1� 107cells=mL. An amount of 800 mL of the aforementioned cell suspension wastaken in the electroporation cuvette (4mm) and 10 mg linearized recombinant vectorharboring EPO gene was added. The cells were then electroporated, using the BTXECM 630 electroporater at a set voltage of 280V and capacitance of 900 mF. Theresulting time constant of electroporation was 12 msec. Transfected cells electropo-rated with only plasmid pUB6=V5-HisB were used as negative control. Transfectedcells were cultured in a culture flask. After 24 hours of growth in the culture flask,cells were trypsinized and 103 cells=well were seeded in three 96 well plates with

200 M. SURENDARBABU AND S. MEENAKSHISUNDARAM

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

10 mg=mL blasticidin selection antibiotic in the medium. The cells were incubated inthe 5% CO2 incubator for 10 days.

Western Blotting

As the transfected cells reached confluency, the supernatant was collected andmixed with sample buffer and boiled. The supernatant was resolved by 12%SDS-PAGE and the transfer was carried out at 20V, 120mA for 2 hours using asemi-dry blotting apparatus. Later, the protein transferred membrane was blockedwith 5% skimmed milk, and then the blot was probed with anti-mouse Epo B-4 anti-body (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by ALP-conjugated anti-mouse IgG. The color development was carried out by using 33 mLof 5-bromo-4-chloro-indolyl phosphate (50mg=mL in diethyl formamide) and 66 mLof nitroblue tetrazolium (50mg=mL in 70% diethyl formamide) in 10mL of detectionbuffer. The reaction was stopped after 15 minutes by washing with PBS.

Quantification of EPO

The EPO amount produced by the CHO K1 clone was quantified by competi-tive ELISA. Commercial EPO (Eprex) was used as standard. Varying volume super-natant was pre-incubated with anti EPO primary antibody. Then, the incubatedcomplex was added to the well-coated EPO antigen. The absorbance was measuredafter adding secondary antibody and the substrate. A decrease in OD was observeddue to the competitive binding.

ERYTHROID COLONY FORMATION ASSAY

The erythroid colony formation assay method was followed per the literature(20). The murine bone marrow derived stem cells was used for the bioactivity assayof EPO. The cells were counted and seeded such that there were 2� 104 cells=well inDMEM medium with 0.9% methylcellulose as a viscous support, 2-mercaptoethanol(10�4M), glutamine (2mM), and 30% FBS in 24 well plates. The aforementionedmedium composition was adapted from Gribaldo et al. (21). Various concentrationsof Standard EPO, 100 mL of culture supernatant containing 150 ng, 90 ng, 60 ng,55 ng, and 120 ng rh-EPO of clones 2, 7, 14, 28, and 32, respectively, were mixedin the medium mixture. The culture was incubated for 48 hours at 37�C in 5%CO2 incubator, thereafter the CFU-E (colony forming units-erythroid) colonies werestained by overlaying 15 mL of freshly prepared freshly prepared benzidine solution(H2O-3% benzidine in 95% acetic acid-30% H2O2¼ 200:30:1). Colonies were countedin the whole area of each well using an inverted microscope.

RESULTS AND DISCUSSION

Expression Vector Design and Construction

The shuttle vector pUB6=V5-HisB possesses the characteristic human ubiqui-tin C promoter (hUbC), which allows high level production of recombinant proteins.

HUMAN UBIQUITIN C PROMOTER BASED EXPRESSION 201

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

The vector allows high-level stable expression and also contains blasticidin resistancegene (BSD) for selection of stable cell-lines. The promoter in this plasmid lacks the 5’flanking end that contains the regulatory part of the promoter and, hence, facilitatesconstitutive expression of rh-EPO. The EPO gene with a 27 amino acid N terminalsignal sequence (583 bp) was amplified from BHK cell line genomic DNA at 57�Cannealing temperature (Fig. 1). This gene was cloned between the HindIII and ApaIrestriction sites in the vector pUB6V5HisB under the control of the ubiquitin pro-moter. The recombinant construct pUB6=V5-HisB-EPO was then transformed toE.coli and the positive colonies were screened in the ampicillin agar plates. The pres-ence of EPO gene was confirmed using lysate PCR and the gene sequence was con-firmed using human ubiquitin C promoter forward and bovine growth hormonereverse primers. The recombinant plasmid was linearized for stable transfectionusing the BglII restriction site.

Qin et al. (22) compared different constitutive promoters (SV40, CMV, UBC,EF1A, PGK, and CAGG) in different mammalian cell types using the GFP reportergene expression. They reported that the commonly used CMV promoter expressedthe proteins at varying levels in different cell types highlighting the importance ofpromoter cell-type combination. They also reported that the UBC promoter is aweak promoter and the expression levels are lower in mammalian cell lines. Byunet al. (23) studied the effect of UBC promoter for expression of IL-2 and GFP inhematopoietic TF-1 and mesenchymal progenitor cells and compared the expressionof this promoter with commonly used CMV promoter. They have observed that forboth the proteins, the expression levels of UBC promoter are higher than the CMVpromoter in the in vitro culture conditions. Spenger et al. (24) also reported that incomparison with the UBC and viral promoters such as CMV, SV40, and RSV in dif-ferent cell lines such as CHO, COS, and so forth, the expression of proteins by the

Figure 1 PCR Amplification of EPO gene from the BHK cDNA using gene specific primers. The 1% agar-

ose gel shows the amplified fragment of EPO gene from the BHK cDNA clone using gene specific primers.

The amplified fragment corresponds tho 583 bp length. Lane 1: 100 bp DNA ladder (Fermentas); Lane 2:

PCR product (EPO). (Color figure available online.)

202 M. SURENDARBABU AND S. MEENAKSHISUNDARAM

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

promoters are cell specific. In this paper we attempted to express EPO using theUbiquitin promoter in the CHO K1 cell line.

Minimum Inhibitory Concentration of Blasticidin forCHO K1 Cell Lines

It is essential to determine the minimum inhibitory concentration (MIC) ofblasticidin for CHO K1 cell lines used in screening the transfectants. To establishMIC, 50% confluent CHO K1 cell lines were treated with blasticidin under normalin vitro conditions. Figure 2 shows the survival of cells on the seventh day. It canbe seen that there is a negative gradation in the number of live cells as the concen-tration of blasticidin in the medium increases. The negative control containing CHOK1 cell lines without blasticidin had grown to confluency, but at 10 mg=mL concen-tration, no viability was observed. Therefore, for screening stable transfectants,10 mg=mL blasticidin concentration was selected.

Transfection of CHO K1 Cell Lines

CHO K1 host cell lines were transfected with a pUB6=V5-HisB shuttle vectorencoding the gene EPO. In order to get more transfected cells with better expressionof recombinant construct, electroporation was used, because it requires fewer amountsof DNA and high transfection efficiency (25). The stable transfectants that showedresistance to the blasticidin antibiotic selection pressure were primarily screened bymicroscopic observation after 7–10 days and obtained 111 single colonies per well.

Figure 2 Blasticidin resistance for CHO K1 cell lines. Blasticidin resistance for CHO K1 cell lines

graphical representation. It can be seen that there is a negative gradation in the number of live cells as

the concentration of blasticidin in the medium increases. The negative control had grown to confluency.

In 10mg=mL concentration, no cell was alive.

HUMAN UBIQUITIN C PROMOTER BASED EXPRESSION 203

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

Screening for Hyper Producers and Expression of EPO

In order to select a high producer clone for increasing the efficiency of theexpression of rh-EPO and to make the screening procedure shorter, stable cloneswere selected on the basis of increasing the selection pressure. In the present study,the selection pressure was increased from 10 to 100 mg=mL blasticidin in a stepwisemanner (25, 50, 75, and 100 mg=mL), which is multiple folds higher than the normalminimum inhibitory constant for CHO K1 cell lines in subsequent passages. Thirtythree colonies showed resistance to the increased selection pressure of 50 mg=mlLblasticidin and with further increase in the antibiotic concentration up to 100 mg=mL, only five colonies showed resistance. The survival capacity of the colonies sub-jected to further increase of 150 mg=mL was nil. The survival of the colonies toincrease in the antibiotic selection pressure is shown in Fig. 3. The decrease in thenumber of colonies with respect to the stepwise increase of blasticidin selectionpressure concentration is shown in Fig. 4.

The presence of EPO gene in these colonies was checked by extracting thegenomic DNA and performing PCR with gene specific primers (Fig. 5). Expressionsof rh-EPO on these five transfectants along with few different lower concentrationresistant transfectants were checked in Hams F12 medium by doing western blotanalysis. In lower resistance transfectants, the expression levels were not detectablewhile in the five higher resistant transfectants, varying levels of expression wereobserved in western blotting of around 35 kDa (Fig. 6A). The expression levels ofrh-EPO in all the five clones were quantified by competitive ELISA (Fig. 6B). Theexpression of rh-EPO was found to vary between 0.55 to 1.5 mg=mL (106 cells=mL).

In order to simplify the screening process and to increase the efficiency in iden-tifying the positive clones with better yields, secondary screening was carried out athigh selection pressure followed by the primary screening with 10 mg=mL. The selec-tion pressure was increased to a blasticidin concentration of 100 mg=mL, whichresulted in a 10-fold higher concentration than the standardized minimum inhibitory

Figure 3 Microscopic observation of Single colony=well resistant to blasticidin. Single colony=well micro-

scopic observation for the positive clones. The microscopic observation showed the colonies resistant to

blasticidin antibiotic in the primary screening. 1–5: Single colony=well microscopic observation of clones

2, 7, 14, 28, and 32; 6: Host cell line (CHO K1)-negative control. (Color figure available online.)

204 M. SURENDARBABU AND S. MEENAKSHISUNDARAM

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

concentration for CHO K1 cell lines. This method results in isolation of highproducer cell lines in a few weeks. Yoshikawa et al. (26) mentioned that in the screen-ing method for transfected CHO=dhfr cell lines based on methotrexate (MTX),

Figure 5 PCR analysis of genomic DNA of blasticidine resistance transfectants. Agarose gel electrophoresis

shows the EPO gene insertion in CHOK1 genome. The PCR amplification performed using CHOK1 genome

as template using gene specific primers. Lane 1: 100BP DNA ladder (Fermentas); Lane 2: positive-EPO gene;

Lane 3, 4, 5, 7, 8: genomic DNA pcr product of clones 14, 2, 28, 32, and 7, respectively; Lane 6: Negative con-

trol—genomic DNA pcr of CHO K1—transfected with the vector alone. (Color figure available online.)

Figure 4 Screening of stable clones by increasing the selection pressure. The colonies resistant to 10mg=mL

were subjected to gradual increase in the blasticidin antibiotic up to 150mg=mL. The graph shows the

decline in the number of resistant colonies. The colonies resistant to 100mg=mL were five showed higher

expression but other resistant colonies showed negligible level of expression. Selection pressure was

increased 10-fold, higher than the standardized minimum inhibitory concentration for CHO K1 to obtain

high expression clones. The sequential increase in the antibiotic concentration results in the high copy

transfectants. (Color figure available online.)

HUMAN UBIQUITIN C PROMOTER BASED EXPRESSION 205

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

increasing the concentration of antibiotic leads to selection of high producer clonesthrough flow cytometry analysis. Normally, an increase in antibiotic concentrationmay lead to an increase in expression or an increase of the gene copy number inthe transfected cell lines genome. However, we used this methodology of increasingthe blasticidin concentration to screen the high producer clones in a shorter time per-iod in a stepwise manner without applying any sophisticated technique. As per themethod we adopted, five transfectants with high antibiotic resistance showed higherlevels of expression, when compared to expression levels of lower resistant transfec-tants. By using the secondary stepwise screening approach, the highly productivetransfectants can be easily separated without using any bio instrumentation techni-ques. The adaptive response of the transfected cell lines for the higher blasticidinselection pressure resulted in a high level of rh-EPO expression.

Figure 6 A. Western blot analysis of expression of Erythropoietin in CHO KI Cell line transfectants.

Western blotting of supernatant of stable clones, pUB6 V5-His B-EPO using anti-EPO monoclonal anti-

body. Lane 1: Protein marker (fermentas); Lane 2: Eprex—commercial positive control; Lane 4–8: Culture

superntants of clones 2, 7, 14, 28, and 32, respectively; Lane 3: Culture superntant (CHO K1-vector alone)

negative control. B.Quantification of EPO in all the five clones by competitive ELISA. The different level

of rh-EPO expressions were observed in the CHO K1 stable transfectants. The expression pattern varies

because of the random integration of the expression cassette in the host cell genome. The high expression

level of 1.5mg=mL rh-EPO was achieved in clone 2. (Color figure available online.)

206 M. SURENDARBABU AND S. MEENAKSHISUNDARAM

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

The cloning of adherent cells is relatively easy and can be carried out for effectiveselection of individual colonies, whereas suspension is difficult to screen; however, suspen-sion helps to increase the cell density (27). Though all the five clones were screened on thesame antibiotic concentration, there is a difference in the production levels and this maybe due to the site of integration in the host chromosome. The major factor in the success-ful expression of any gene from stable cell line is dependent on the transcription rate,which, in turn, depends on the site of integration of linearized plasmid DNA in thechromosomal DNA of the host. Obtaining hyper-expressing clones is based on randomintegration of plasmid DNA without disrupting the important regulatory elements (28).

Production of recombinant erythropoietin have been reported in different cell linessuch as COS cells (12, 29, 30), BHK cells (29, 31), CHO cells (32–35), PSI -2 cells (31), andinsect cell lines (11). EPO is a glycoprotein hormone, and it is important for the protein toundergo glycosylation. Schorpp et al. (36) have expressed transgenes in mice using humanubiquitin C promoter and have found that this promoter helps in post translational modi-fication of various proteins. In this paper, we reported the production of erythropoietinwith UBC promoter CHO cell line combination. Here, we are able to get moderateexpression in the normal expressionmedium. Further optimization and suspension cultureconditions might improve the yield of this recombinant protein in this system.

EPO activity is tested by the formation of erythroid colonies. EPO expressed in thesupernatant of stably transfected CHO-K1 cells were assayed for activity. CommercialEPO (EPREX) was used as positive control. The CFU E by the murine bone marrowderived stem cells with standard EPO and rh-EPO were tested and showed maximum col-ony formation in the supernatant of clone 2 in the benzidine staining after 48 hours (Fig. 7).

Figure 7 Erythroid colony formation assay. A. The effect of standard EPO in murine derived bone marrow

stem cells, resulted in increase in the formation of erythroid colonies with rise in the concentration of EPO.

B.The culture supernatant of all the 5 clones were tested for CFU-E colonies in the cells and showed a

response for bioactivity. The clone 2 showed a higher number of erythroid colonies with respect to the

rh-EPO expression level. C. Shows photos of benzidine stained erythroid colonies obtained on stimulation

with the supernatants of stably transfected CHO-K1 cells and standard EPO after 48 hours.

HUMAN UBIQUITIN C PROMOTER BASED EXPRESSION 207

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

CONCLUSION

The results showed that the human ubiquitin C promoter CHO cell linecombination produced a moderate expression of rh-EPO. The analysis on bioactivityreported that the recombinant protein displayed erythroid cluster forming activity,and the screening approach was found to be effective and simple for the identifi-cation of high producer clones in a short time span.

REFERENCES

1. Jayapal KP, Wlaschin KF, Yap MGS, Hu WS. Recombinant protein therapeutics fromCHO cells-20 years and counting. Chem Eng Prog 2007; 103(10):40–47.

2. Puck TT. Development of the Chinese Hamster Ovary (CHO) Cell, Molecular CellGenetics. New York: John Wiley and Sons, 1985:37–64.

3. Weibe ME. A Multifaceted Approach to Assure that Recombinant tPA is Free ofAdventitious Virus, Advances in Animal Cell Biology and Technology. London:Butterworth-Heinemann, 1989:68–71.

4. Elliott S. Enhancement of therapeutic protein in vivo activities through glycoengineering.Nat Biotechnol 2003; 21(4):414–421.

5. Koury MJ, Bondurant MC. The molecular mechanism of erythropoietin action. EurJ Biochem 1992; 210:649–663.

6. Ng T, Marz G, Wood TL, Macdougall I. Recombinant erythropoietin in clinical practice.Postgrad Med J 2003; 79(933):367–376.

7. Boissel JP. Erythropoietin structure-function relationships: Mutant proteins that test amodel of tertiary structure. J Biol Chem 1993; 268(21):15983–15993.

8. Bill RM, Winter PC, McHale CM, et al. Expression and mutagenesis of recombinanthuman and murine erythropoietins in Escherichia coli. Biochim Biophys Acta 1995;

261(1):35–43.9. Celik E, Calik P, Oliver SG. Fed-batch methanol feeding strategy for recombinant protein

production by Pichia pastoris in the presence of co-substrate sorbitol. Yeast 2009;

26(9):473–484.10. Quelle FW, Caslake LF, Burkert RE, Wojchowski DM. High-level expression and puri-

fication of a recombinant human erythropoietin produced using a baculovirus vector.Blood 1989; 74:652–657.

11. Long DL, Doherty DH, Eisenberg SP, et al. Design of homogeneous, monopegylated ery-thropoietin analogs with preserved in vitro bioactivity. Exp Hematol 2006; 4(6):697–704.

12. Jacobs K, Shoemaker C, Rudersdorf R, et al. Isolation and characterization of genomicand cDNA clones of human erythropoietin. Nature 1985; 313:806–809.

13. Kim SY, Lee JH, Shin HS, Kang HJ, Kim YS. The human elongation factor 1 alpha(EF-1 [alpha]) first intron highly enhances expression of foreign genes from the murinecytomegalovirus promoter. J Biotechnol 2002; 93(2):183–187.

14. Yoon SK, Kim SH, Song JY, Lee GM. Biphasic culture strategy for enhancing volumetricerythropoietin productivity of Chinese hamster ovary cells. Enzyme Microbial Technol2006; 39(3):362–365.

15. Wiborg O, Pedersen MS, Wind A. The human ubiquitin multigene family: some genescontain multiple directly repeated ubiquitin coding sequences. EMBO J 1985; 4(3):755–759.

16. Takeuchi S, Hirayama K, Ueda K, Sakai H, Yonehara H, Blasticidin S. A new antibiotic.J Antibiot (Tokyo) 1958; 11(1):1–5.

17. Yamaguchi H, Yamamoto C, Tanaka N. Inhibition of protein synthesis by blasticidin,studies with cell-free systems from bacterial and mammalian cells. J Biochem 1965;

57(5):667–677.

208 M. SURENDARBABU AND S. MEENAKSHISUNDARAM

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013

18. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold SpringHarbor: Paperback by Laboratory Press. 2001.

19. Takagi J. Establishment of Stable Transfectant of CHO Lec Cells. Springer Lab Protocols,Boston, 2000.

20. Terszowski G, Waskow C, Conradt P, et al. Prospective isolation and global gene expressionanalysis of the erythrocyte colony-forming unit (CFU-E). Blood 2005; 105:1937–1945.

21. Gribaldo L, Malerba I, Collatta A, Casati S, Pessina A. Inhibitin of CFU-E=BFU-E by3’-azido-3’deoxythymidine, chlorpropamide, and protoporphirin IX zinc(II). A compari-son between direct exposure of progenitor cells and long term exposure of bone marrowcultures Toxicol Sci 2000; 58:96–101.

22. Qin JY, Zhang L, Clift KL, et al. Systematic comparison of constitutive promoters andthe doxycycline inducible promoter. Plos One 2010; 5(5):e10611.

23. Byun HM, Suh D, Jeong Y. Plasmid vectors harboring cellular promoters can induceprolonged gene expression in hematopoietic and mesenchymal progenitor cells. BiochemBiophy Res Commun 2005; 322(2):518–523.

24. Spenger W, Ernst JP, Condreay TA, Kost R, Grabherr, R. Influence of promoter choiceand trichostatin – A treatment on expression of baculovirus delivered genes in mammaliancells. Protein Expr Purif 2004; 38:17–23.

25. Chu L, Hayakawa H, Berg P. Electroporation for the efficient transfection of mammaliancells with DNA. Nucleic Acids Res 1987; 15(3):1311–1326.

26. Yoshikawa T. Flow cytometry: an improved method for the selection of high productivegene amplified CHO cells using flow cytometry. Biotechnol Bioeng 2001; 74(5):435–442.

27. Davis JM. Basic cell culture-A Practical approach. 2nd ed. Oxford University Press,New York, 2002.

28. Wurm FM. Production of recombinant protein therapeutics in cultivated mammaliancells. Nat Biotech 2004; 22:1393–1398.

29. Powell S, Berkner KL, Lebo RV, Adamson JW. Human erythropoietin gene: High levelexpression in stably transfected mammalian cells and chromosome localization. Proc NatlAcad Sci USA 1986; 83:64–65.

30. Wojchowski DM, Sue JM, Sytkowski AJ. Site-specific antibodies to human erythropoie-tin: Immunoaffinity purification of urinary and recombinant hormone. Biochim BiophysActs 1987; 9:13–17.

31. Goto M, Akai K, Murakami A, et al. Production of recombinant human erythropoietin inmammalian cells: Host-cell dependency of the biological activity of the cloned glyco-protein. Biotechnol 1988; 6:67.

32. Lin FK, Lin CH, Browne JK, et al. Cloning and expression of the human erythropoietingene. Proc Natl Acad Sci USA 1985; 82:7580.

33. Davis JM, Arakawa T, Strickland TW, Yphantis DA. Characterization of recombinanthuman erythropoietin produced in Chinese hamster ovary cells. Biochem 1987; 26:2633.

34. Recny MA, Scoble HA, Kim Y. Structural characterization of natural human urinary andrecombinant DNA-derived erythropoietin. J Biol Chem 1987; 262:17156.

35. Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietinexpressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J Biol Chem1987; 262:12059.

36. Schorpp M, Jager R, Schellander K, et al. The human ubiquitin C promoter directs highubiquitous expression of transgenes in mice. Nucleic Acids Res 1996; 24:1787–1788.

HUMAN UBIQUITIN C PROMOTER BASED EXPRESSION 209

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

04:

34 1

0 D

ecem

ber

2013