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Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

Orlova NA, Kovnir SV, Hodak JA, Vorobiev II, Gabibov AG, Skryabin KG - BMC Biotechnol. (2014)

Bottom Line: Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other.The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1.The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Mammalian Cell Bioengineering, Centre "Bioengineering", Russian Academy of Sciences, 60-letija Oktyabrya 7, Moscow 117312, Russia. ptichman@gmail.com.

ABSTRACT

Background: Establishing highly productive clonal cell lines with constant productivity over 2-3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure.

Results: We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative.

Conclusions: The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

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Distribution of the eGFP expression levels in cell populations as determined by FACS analysis. Codes for the corresponding cell populations are the same as in Figure 3. First number after the cell population code: mean level of eGFP in the sample; second number: mean level of eGFP in the upper 10% of the eGFP-positive cells.
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Figure 5: Distribution of the eGFP expression levels in cell populations as determined by FACS analysis. Codes for the corresponding cell populations are the same as in Figure 3. First number after the cell population code: mean level of eGFP in the sample; second number: mean level of eGFP in the upper 10% of the eGFP-positive cells.

Mentions: Heterologous expression of a functionally active target protein often requires co-expression of a small number of protein processing enzymes. For example, the blood clotting factor IX expression systems used with CHO or BHK cells rely on co-expression of the signal protease PACE/furin [17] and the vitamin-K recharging enzyme, VKORC1 [18]. Generally, the expression levels of such “helper” proteins should be lower than that of the target protein, but of comparable magnitude. If the target protein is coded by a plasmid bearing a DHFR selection marker, helper proteins may be coded by plasmids with the same structure, but bearing antibiotic resistance markers. We have tested resistance markers for three widely used antibiotics, G418 (a neomycin analogue), zeocin, and hygromycin, in the EEF1A-based expression vector, which was modified by removing the IRES fragment and the DHFR open reading frame from the p1.1 plasmid, and insertion of the corresponding antibiotic resistance genes outside of the EEF1A gene flanking areas and controlled by a separate SV40 promoter. The resulting vectors, denoted p1.2-Neo, p1.2-Zeo, and p1.2-Hygro, were used for insertion of the eGFP protein ORF. All three resulting plasmids showed similar transient transfection efficiencies in CHO DG44 cells (19–24% by electroporation), and the resulting cell populations were used to generate stably transfected cell populations in the suspension culture under variable selection pressures for each antibiotic used. The control plasmids p1.1eGFP and p1.1(EBVTR-)eGFP were transfected using the same procedure and stably transfected cell populations were generated by suspension cultivation in the presence of 200 nM MTX. The populations obtained were examined to determine the proportion of eGFP-expressing cells and eGFP levels in cell lysates (Figure 3). We found that for all three selection markers at all levels of drug selection pressure the resulting cell populations contained more than 75% of eGFP-positive cells. For the hygromycin and MTX resistance markers, less than 5% of the cells were eGFP-negative. The level of eGFP in the cell lysates was maximal for hygromycin selection, peaking as 8.9% of the total cellular protein with 0.5 mg/ml of hygromycin. In contrast, eGFP levels in the polyclonal cell populations obtained from transfection with p1.1eGFP or p1.1(EBVTR-)eGFP were much lower at 1.9% and 1.0%, respectively; however, eGFP expression levels for the p1.1 vector could potentially increase by eight-fold using the MTX-driven target gene amplification described above.We also measured the intracellular eGFP distribution in polyclonal cell populations using FACS (Figure 5). Virtually no cells were eGFP-negative with DHFR and hygromycin selection markers, whereas with the neomycin resistance gene the level of eGFP-negative cells was inversely proportional to the concentration of G418 used. The mean eGFP level for the upper 10% of the eGFP-positive cells was not dependent on the antibiotic concentration for neomycin and zeocin selection, whereas with hygromycin selection the mean eGFP level was higher at higher antibiotic concentrations.


Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

Orlova NA, Kovnir SV, Hodak JA, Vorobiev II, Gabibov AG, Skryabin KG - BMC Biotechnol. (2014)

Distribution of the eGFP expression levels in cell populations as determined by FACS analysis. Codes for the corresponding cell populations are the same as in Figure 3. First number after the cell population code: mean level of eGFP in the sample; second number: mean level of eGFP in the upper 10% of the eGFP-positive cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4067061&req=5

Figure 5: Distribution of the eGFP expression levels in cell populations as determined by FACS analysis. Codes for the corresponding cell populations are the same as in Figure 3. First number after the cell population code: mean level of eGFP in the sample; second number: mean level of eGFP in the upper 10% of the eGFP-positive cells.
Mentions: Heterologous expression of a functionally active target protein often requires co-expression of a small number of protein processing enzymes. For example, the blood clotting factor IX expression systems used with CHO or BHK cells rely on co-expression of the signal protease PACE/furin [17] and the vitamin-K recharging enzyme, VKORC1 [18]. Generally, the expression levels of such “helper” proteins should be lower than that of the target protein, but of comparable magnitude. If the target protein is coded by a plasmid bearing a DHFR selection marker, helper proteins may be coded by plasmids with the same structure, but bearing antibiotic resistance markers. We have tested resistance markers for three widely used antibiotics, G418 (a neomycin analogue), zeocin, and hygromycin, in the EEF1A-based expression vector, which was modified by removing the IRES fragment and the DHFR open reading frame from the p1.1 plasmid, and insertion of the corresponding antibiotic resistance genes outside of the EEF1A gene flanking areas and controlled by a separate SV40 promoter. The resulting vectors, denoted p1.2-Neo, p1.2-Zeo, and p1.2-Hygro, were used for insertion of the eGFP protein ORF. All three resulting plasmids showed similar transient transfection efficiencies in CHO DG44 cells (19–24% by electroporation), and the resulting cell populations were used to generate stably transfected cell populations in the suspension culture under variable selection pressures for each antibiotic used. The control plasmids p1.1eGFP and p1.1(EBVTR-)eGFP were transfected using the same procedure and stably transfected cell populations were generated by suspension cultivation in the presence of 200 nM MTX. The populations obtained were examined to determine the proportion of eGFP-expressing cells and eGFP levels in cell lysates (Figure 3). We found that for all three selection markers at all levels of drug selection pressure the resulting cell populations contained more than 75% of eGFP-positive cells. For the hygromycin and MTX resistance markers, less than 5% of the cells were eGFP-negative. The level of eGFP in the cell lysates was maximal for hygromycin selection, peaking as 8.9% of the total cellular protein with 0.5 mg/ml of hygromycin. In contrast, eGFP levels in the polyclonal cell populations obtained from transfection with p1.1eGFP or p1.1(EBVTR-)eGFP were much lower at 1.9% and 1.0%, respectively; however, eGFP expression levels for the p1.1 vector could potentially increase by eight-fold using the MTX-driven target gene amplification described above.We also measured the intracellular eGFP distribution in polyclonal cell populations using FACS (Figure 5). Virtually no cells were eGFP-negative with DHFR and hygromycin selection markers, whereas with the neomycin resistance gene the level of eGFP-negative cells was inversely proportional to the concentration of G418 used. The mean eGFP level for the upper 10% of the eGFP-positive cells was not dependent on the antibiotic concentration for neomycin and zeocin selection, whereas with hygromycin selection the mean eGFP level was higher at higher antibiotic concentrations.

Bottom Line: Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other.The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1.The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Mammalian Cell Bioengineering, Centre "Bioengineering", Russian Academy of Sciences, 60-letija Oktyabrya 7, Moscow 117312, Russia. ptichman@gmail.com.

ABSTRACT

Background: Establishing highly productive clonal cell lines with constant productivity over 2-3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure.

Results: We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative.

Conclusions: The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

Show MeSH
Related in: MedlinePlus