Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO)
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Background and Aims
Erythropoietin (EPO) is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO) cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7) to produce recombinant EPO.
Materials and methods
Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV) promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO.
Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (P < 0.05) of EPO was observed in the medium from Huh-7 cell line.
Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.
KeywordsEPO erythropoietin; CHO Chinese hamster ovary cell line; Huh-7 Human hepatoma cell line; PCR polymerase chain reaction
EPO is an essential growth factor required for the proliferation and differentiation of the stem cells that produce red blood cells . Structurally, EPO is a glycoprotein contains approximately 40% carbohydrate . These carbohydrate structures are very essential for many biological properties like pharmacokinetics, secretion, stability, receptor recognition and antigenicity, protein conformation and biological activity .
Kidney is the main production unit of EPO in normal person . Any damage to kidney tissues abolishes the EPO secretion from kidney thus causing anemia in renal patients. Recombinant DNA technology has enabled manufacture of the recombinant human EPO (rHuEPO) to use as a drug. The initial findings with the use of rHuEPO for the treatment of anemia were so inspiring that it was licensed to use as therapy within three years of its availability . Now the drug is amongst the top selling pharmaceutical product worldwide and considered applicable for a variety of disorders such as anemia associated with renal failure, hepatitis C infection, cancer, human immunodeficiency virus infections, and cardiovascular disease .
The glycosylation pattern of protein is affected by several parameters including the protein structure , host system used to produce glycoprotein  and the culture conditions . Among all these parameters, host system greatly affects the glycosylation pattern, thus selection of appropriate host system is very crucial to produce the therapeutic glycoprotein. Among the prokaryotic and eukaryotic expression systems only mammalian cell lines have the capacity to carry out proper glycosylation. Currently, the mammalian system used to produce the rHuEPO is CHO cell line  associated with some disadvantages e.g. it is not able to control the human like glycosylation. EPO produced from CHO cells contains N-glycolylneuraminic acid , but humans lack the pathway for the synthesis of N-glycolylneuraminic acid , thus recombinant EPO that contain N-glycolylneuraminic acid are subjected to clearance by anti-N-glycolylneuraminic acid antibodies present in human serum . Therapeutic use of such glycoproteins may cause undesirable responses that can affect the efficacy of the treatment. So the use of human cell line for therapeutic glycoproteins production is unavoidable. Human-cell based expression system can provide some unique characteristics to biopharmaceutics including lower immunogenicity, greater biological activity and increased half life.
With the trend to introduce the human-cell-based expression system to synthesize the biopharmaceutical products, several human cell lines have been proposed e.g. Human embryonic kidney (HEK293) and human fibro-sarcoma (HT-1080) cell line . In the present study, we have used Huh-7 cell line for the first time for expression study of EPO gene in comparison to CHO cell line. Previously this cell line was used for the expression study of insulin [15, 16]. Huh-7 cell line is derived from epithelial cells of human liver. We hypothesized that EPO produced from human liver cell line would be properly folded and glycosylated, as EPO is mainly produced by liver cells during the fetal stage .
Materials and methods
Construction of vectors for erythropoietin gene expression
Signal sequence and Kozak addition
In another approach six nucleotides of Kozak sequence (GCCACC), was also added before the start codon of EPO-cDNA construct by using the primer K as forward and primer R as reverse primers (Table 1). The size of this fragment was 594 bp including the Xho 1 and BamH 1 restriction sites at 5' and 3' end respectively. PCR-product was digested with Xho1 and BamH1 and used for ligation into plasmid pcDNA3.1/zeo the resulting plasmid was named as pcDNA3.1-K-SS-EPO (Figure 2A). Following transformation and subsequent plasmid purification, of randomly selected clones, restriction digestion and sequencing analysis was performed using Big Dye chain termination reaction on an ABI 3100 DNA sequencer (Applied BioSystem) to verify the cloning of EPO-cDNA constructs in expression vector.
Huh-7 cell line was maintained in 75 cm2 culture flasks (Iwaki, Japan) containing DMEM (Sigma Aldrich, USA) supplemented with 100 μg/ml penicillin/streptomycin, and 10% FBS as complete culture media (CCM) (Sigma Aldrich, USA), at 37°C with 5% CO2. The CCM was renewed every third day and were passaged every 4-5 days. Viable cells were counted using 0.5% trypan blue (Sigma Aldrich, USA). Similarly CHO cell line was maintained in DMEM Ham's F12 (Sigma Aldrich, USA) and maintained as described for Huh-7 cells.
Approximately 3.5 × 105 CHO cells were seeded into 60 mm plates and cultured in CCM until 60-80% confluent prior to transfection. 8 μg of pcDNA3.1-EPO, pcDNA3.1-SS-EPO and pcDNA3.1-K-SS-EPO was transiently transfected in CHO cell line in serum-free media using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. After 6 hours incubation at 37°C in 5% CO2, cells were washed with 1x PBS and CCM was added to the cells. Cells were harvested at 24 hours post-transfection to analyze the EPO expression at mRNA and protein level. Similarly the Huh-7 cells were transfected as described above.
RT-PCR (Reverse transcriptase PCR)
For EPO gene expression analysis, total RNA from Huh-7 and CHO cells transiently transfected with mock, EPO expression plasmids was extracted using Purescript RNA isolation kit (Gentra, USA) according to manufacturer's protocol. cDNA was synthesized using 1 μg of total RNA with SuperScript III enzyme (Invitrogen, USA). EPO gene expression analysis was carried out using RT-PCR on ABI 2700 PCR machine using gene specific primers. PCR conditions were as follow: 95°C, 30 sec; 57°C, 30 sec and extension at 72°C for 40 sec for 30 cycles. GAPDH was used as an internal control.
ELISA (Enzyme Liked Immunosorbent assay)
For intra-cellular expression analysis of EPO, cells were harvested with ProteoJET mammalian cell lysis reagent (Fermentas, Canada) and for extra-cellular EPO expression supernatant was collected after 24 hours of transfection. Intra and extra-cellular EPO expression analysis was performed through ELISA (DRG, USA) according to manufacturer's protocol. Total 50 μg of intra-cellular protein and 200 μl of media for extra-cellular EPO expression analysis were subjected to ELISA. Absorbance was read at 450 nm on ELISA plate reader to determine the concentration of EPO in samples. The intensity of the yellow color developed was directly proportional to the concentration of EPO.
Mature erythropoietin is secreted into the medium, so supernatant was collected for comparative extra-cellular EPO expression analysis from CHO and Huh-7 cells, 24 hours post transfection. Samples and commercially available recombinant human erythropoietin (as a positive control) were subjected to electrophoresis on 12% SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane using manufacturer's protocol (Bio-Rad, USA). Blots were incubated with specific-monoclonal antibodies of EPO (Santa Cruz Biotechnology, Inc, USA). Protein expression was evaluated using chemiluminescence detection kit (Sigma Aldrich, USA).
Results and discussion
Construction of secretory plasmid
Effect of Kozak sequence on erythropoietin expression
Comparative expression analysis of EPO in CHO and Huh-7 cell line
Our results showed that Huh-7 cells have the potential to secrete the comparable level of EPO to the CHO cell line which is already being used for commercial production of erythropoietin. Although, CHO cell line has several advantages including high growth rate and high productivity, but human cell-based expression is expected to produce EPO with post-translational modifications similar to their natural counterpart as some sugar tarnsfering enzymes are not present in CHO cell line e.g. α2-6 sialyltransferase and α1-3/4 fucosyltransferase .
In many studies people have used Huh-7 cell line as model system for expression studies of human proteins [25, 26, 27, 28]. In the present study, Huh-7 cell line has shown the potential to produce the glycosylated EPO protein as an initial finding. However, to produce EPO at commercial level using Huh-7 cell line further characterization should be undertaken to determine the exact extent of sialylation, as it is reported that increased sialylation of EPO has been shown to increase its circulatory half-life. And this could be done through high pH anion exchange chromatography and isoelectric focusing.
Further studies to evaluate the bioactivity, up scalability and compatibility of Huh-7 cells with bioreactor are also needed. These findings will not only assist the biopharmaceutical industry by providing a human-cell-based expression system to produce recombinant glycosylated protein but, is also a preliminary step to make use of liver cells to deliver the EPO gene in chronically infected renal patients to cure anemia.
HK, BI and MTS are Ph.D. scholars in discipline of Molecular Biology at CEMB, University of the Punjab, Lahore, WA (M Phil Chemistry) and SG (MSc Biochemistry) are Research Officers; ZI is Ph.D. scholars at Radboud University Nijmegen Medical Center, Nijmegen, Netherlands; ZN (Biological Sciences) is Associate Professor in Biochemistry & Molecular Biology department, University of Miami, USA; while SH (PhD Molecular Biology) is Principal Investigator at CEMB, University of the Punjab, Lahore.
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