Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression

Abstract

Drosophila melanogaster S2 cells were co-transfected with plasmid vectors containing the enhanced green fluorescent protein gene (EGFP), under the control of metallothionein promoter (pMt), and the hygromycin selection gene, in view of establishing parameters for optimized gene expression. A protocol of transfection was worked out, leading after hygromycin selection, to ∼90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO₄) induction. As analyzed by confocal microscopy, S2MtEGFP cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of fluorescence among the EGFP expressing cells. Spectrofluorimetry kinetic studies of CuSO₄ induced S2MtEGFP cells showed the EGFP expression at 510 nm as soon as 5 h after induction, the fluorescence increasing progressively from this time to attain values of 4.6 × 10⁵ counts/s after 72 h of induction. Induction with 700 μM of CuSO₄ performed at the exponential phase of the S2MtEGFP culture (10⁶ cells/mL) led to a better performance in terms of cell growth, percent of fluorescent cells and culture intensity of fluorescence. Sodium butyrate (NaBu) treatment of CuSO₄ induced S2MtEGFP cell cultures, although leading to a loss of cell culture viability, increased the percent of EGFP expressing cells and sharply enhanced the cell culture fluorescence intensity. The present study established parameters for improving heterologous protein expression in stably transfected Drosophila S2 cells, as assessed by the EGFP expression.

Introduction

Drosophila melanogaster Schneider 2 (S2) cells have become increasingly utilized over the past few years for the expression of heterologous proteins. High levels of protein expression with pharmacological and biotechnological interest can be achieved using the Drosophila expression system (DES) procedure whereby a plasmid encoding a gene of interest is co-transfected with a plasmid encoding resistance to hygromycin B. After three weeks of selection this system, using Drosophila promoters, is able to generate a stable polyclonal cell line processing up to 1,000 gene copies per cell. Proteins of prokaryotic, eukaryotic and viral origin have been expressed in this system showing to be appropriately processed and biologically active (Angelichio et al. 1991; Culp et al. 1991; Han 1996; Li et al. 1996; Delm et al. 1999; Nilsen and Castellino 1999; Lee et al. 2000; Hill et al. 2001; Shin et al. 2003; Perret et al. 2003; Chang et al. 2005). Based on these evidences DES has an important potential as a gene expression system.

Nevertheless, in view of higher expression, some bioprocess parameters have to be optimized. In this context, the transfection procedure, aiming the introduction of foreign DNA into cells, is an important step. The calcium phosphate method has been recommended for gene transfection of S2 cells (Van der Straten et al. 1989) but procedures based on liposome reagent have been reported to be more efficient (Han 1996; Park et al. 1999; Shin and Cha 2002)

Besides the metallothionein promoter induction by copper sulfate (CuSO₄) (Huang et al. 2004) it has been studied the enhancement of gene expression by agents, such as the sodium butyrate (NaBu), a histone deacetylase inhibitor that lead to chromatin exposure and allow the transcriptional factors binding and increased gene expression (Dorner et al. 1989; Li and Li 2006; Zhao et al. 2006; Ping et al. 2006; Song et al. 2006).

The heterologous gene encoding the enhanced green fluorescent protein (EGFP), a mutant from jellyfish Aquorea Victoria (Ward 2006; Phillips 2006), has been proved to be very helpful for the estimation of gene expression in several systems, becoming very attractive as a reporter gene expression (Shin and Cha 2002; Shin et al. 2003; Ward and Lippincott-Schwartz 2006; Hazelrigg and Mansfield 2006). This protein is a 238-amino acid polypeptide that undergoes an intramolecular cyclization to give a brightly fluorescent molecule. It remains remarkably stable in the presence of many denaturants and proteases, as well as over a broad range of pH and temperature (Ward 2006).

We report here in this study an optimization of EGFP stable transfection and inducible conditions for expression using the S2 cells. A protocol for EGFP stable transfection was established by using a lipotransfection method taking into account the cell density, the ratio of DNA concentration and the amount of cellfectin reagent. Conditions for inducible EGFP expression were established by using the metallothionein promoter and CuSO₄ and NaBu as expression inducers. We have evaluated the kinetics cell growth and EGFP expression in a stable transfected, hygromycin selected, polyclonal S2 cell line by confocal microscopy, flow cytometry, and spectrofluorimetry. Our data point to optimized experimental conditions for heterologous protein expression by Drososphila melanogaster S2 cells.

Materials and methods

Cell line, plasmids and enzymes

Drosophila melanogaster Schneider 2 cells (Invitrogen) were grown at 28 °C under normal atmosphere in T-25 flasks in SF900-SFMII medium (Invitrogen). For the EGFP expression, we used the pMT/BiP/V5-His (3.6 KB) expression vector (Invitrogen) which has a metallothionein promoter (pMt) for heavy metals inducible expression. It contains also a sequence of C-terminal peptide containing V5 epitope and a polyhistidine (6× His) tag, besides the elements (pUC ori and ampicillin) for replication in bacteria. For selection of co-transfected cells, we used the pCoHygro (Invitrogen) selection vector containing the hygromycin resistance gene. This gene is under control of the constitutive Drosophila copia 5′LTR promoter. E. coli DH5α was used as a primary host for constructing and propagating plasmids. E. coli were grown and maintained in LB medium containing 50 μg/mL of ampicillin. DNA restriction enzymes and ligases were used according to manufacturer’s instructions (Promega and Fermentas).

Recombinant plasmid construction

cDNA encoding the EGFP was originally isolated from pGie1EGFP plasmid (kindly provided by Dr. Bergmann Morais from the Brasilia University, Brasília, Brazil). The fragment was isolated from electrophoresis gel of pGeie EGFP digested with Apa I and Not I restrictions enzymes (Fig. 1). The fragment was extracted from the gel and purified using a commercial kit (Promega). Cloning was done according to standard techniques (Sambrook and Russel 2001) and the construct (pMtEGFP) was sequenced to check for in-frame insertion.

Fig. 1.

Schematic construction of pMtEGFP vector

Cell culture and transfection

Drosophila S2 cells were grown at 28 °C in SF900-SFMII medium and co-transfected with pMtEGFP and pCoHygro using cellfectin reagent (Invitrogen) for stable expression. Both plasmids (pMtEGFP and pCoHygro) at a ratio of 20:1 and cellfectin reagent (20 μL) were diluted separately in 500 μL of SF900-SFMII medium and then mixed for 15 min at room temperature, constituting the transfection solution (Table 1).

Table 1.

Optimized protocol for transfection of Drosophila S2 cells

Parameters	Established transfection protocol
S2 cell concentration	5 × 106 cells/ T-25 flasks
Ratio expression/selection vectors	20:1
Total DNA	2 μg
Cellfectin® reagent	20 μg
SF900 SFMII medium (dilution)	500 μl
Incubation time (complex formation)	15 min RT
Incubation time (transfection)	5 h

5 × 10⁶ cells were seeded in T-25 flasks and 24 h after the medium was discarded and 1 mL of transfection solution added. After 5 h of incubation with slowly agitation at room temperature, 4 mL of SF900-SFMII medium was added. The cells were incubated for 2 days and the medium replaced with 600 μg/mL of hygromycin B for selection. Hygromycin B resistant cells were then selected as stable polyclonal populations.

Kinetics experiments of EGFP expression by S2MTEGFP cells were performed upon CuSO₄ induction (500 or 700 μM) and/or NaBu (5 mM) treatment of cell cultures performed in 100 mL shake flasks with 20 mL of SF900-SFMII medium incubated at 100 rpm and 28 °C. The cell seeding was the same for all experiments (5 × 10⁵ cells per mL). Cell concentration was determined by viable cells (trypan blue exclusion) counting in hematocytometer.

Confocal microscopy

For confocal EGFP detection, CuSO₄ induced S2MTEGFP cells were scanned using the 488 nm laser line and light emitted between 500 and 600 nm was collected. Images were collected on a META LSM 510 laser scanning confocal microscope equipped with a 63.0 × 1.2 W objective (Carl Zeiss Ltda). The same settings for image acquisition and processing have been applied for all samples to allow comparison of the fluorescence intensities among different samples

Flow cytometry

After induction with the indicated amounts of CuSO_4, 400 μL of S2MTEGFP cells (10⁶ cells) growing in suspension were analyzed by flow cytometry using a FACS-CALIBUR (Becton Dickinson). EGFP fluorescence was excited at 488 nm and emission was measured with a 530/30 nm band pass filter. S2 cells were used as a control for auto fluorescence and to set the gate for EGFP positive cells. 10.000 events/cells were gated from the 400 μL samples. The geometric mean values were used to compare the intensity of fluorescence in different samples as well as the percentage of fluorescent cells.

Spectrofluorimetry

Cell suspensions were analyzed in spectrofluorimeter PTI for fluorescence measurements with a 4 nm band pass. Cell cultures prepared in shake flasks were induced with 700 μM of CuSO₄ and samples (10⁷ cells) were harvested at different times post induction and washed with PBS (1×). The cell suspension (1 mL) was evaluated for fluorescence emission at 490–750 nm when excited with blue light (470 nm).

Statistical analysis

Data were analyzed by the variance F test and statistical differences among values were established by the Tukey test with a significance level of 5%.

Results

EGFP expression vector construction and transfection in S2 cells

We have generated an expression vector encoding the EGFP cDNA by cloning the EGFP fragment into a pMt expression vector in Apa I/Not I cloning sites (Fig. 1). The EGFP gene, under control of the inducible Drosophila metallothionein gene promoter, was inserted in a Drosophila expression vector. This vector was co-transfected with a Drosophila selection vector (carrying hygromicin B resistance gene) into Drosophila cells and a population was selected for their resistance to hygromicin B.

For the co-transfection of pMtEGFP and pCOHygro into S2 cells, we have developed a transfection protocol (Table 1) which allowed us to select a stable polyclonal cell population (S2MtEGFP) with hygromycin B after 2 weeks of culture.

EGFP expression upon CuSO₄ induction

Figure 2 shows the EGFP expression and cell growth of S2MtEGFP cells induced with 500 or 700 μM of CuSO₄ at day 3 of culture. We observed that after 2 days of induction, 700 μM of CuSO₄ were able to induce the EGFP expression in 80% of the cells, in contrast to 500 μM of CuSO₄ that induced the EGFP expression in only 60% of cells. Nevertheless, after 5 days of induction both CuSO₄ concentrations led to EGFP expression in about 85% of cells. The fluorescence intensity also increased with the CuSO₄ concentration and at day 5 of induction we had an intensity of fluorescence of 100 units for cultures induced with 500 μM of CuSO₄ and 142 units for those induced with 700 μM of CuSO₄. The cell growth, at the exponential phase, was shown to be comparable for control or CuSO₄ induced cultures, indicating that the CuSO_4, at these concentrations, had no influence on the cell growth (1.5 to 2 × 10⁷ cells/mL). When the CuSO₄ induction was performed together with cell seeding at the beginning of the culture we have observed a significant decrease in the cell multiplication as well as lower expression levels of EGFP as evaluated by the number of fluorescent cell or the intensity of fluorescence of the cell population (data not shown).

Fig. 2.

Kinetics of cell growth (A), % of fluorescent cells (B) and fluorescence intensity (C) of S2 and S2MtEGFP cells induced with 500 or 700 μM of CuSO₄ at day 3 of culture. •, S2MtEGFP cells + 700 μM of CuSO₄; ■, S2MtEGFP cells + 500 μM of CuSO₄; ♦, S2 cells; ○, S2MtEGFP cells. Arrows indicate the time of induction at day 3. Results are the average of at least three independent experiments. ^*: statistically different

Confocal analysis of CuSO₄ induced S2MtEGFP cells showed an heterogeneous EGFP expression among the cell population (Fig. 3A). Kinetics of EGFP expression as assessed by flow cytometry analysis of S2MtEGFP cells (Fig. 3B) showed in a non-induced S2MtEGFP cell population a low percent of fluorescent cells (8%) which sharply increased upon CuSO₄ induction attaining high levels (88%) at day 5 of induction. By spectrofluorimetry we could better examine the time response of S2MtEGFP cells upon CuSO₄ induction, by analyzing the fluorescence intensity expressed by the cell population in early times after induction. Figure 4 shows that specific fluorescence at 510 mm could be already detected as early as 5 h after induction, showed significant levels at 24 h and increased progressively to attain high levels at 72 h.

Fig. 3.

S2MtEGFP cell culture as evaluated by confocal microscopy (A) and flow cytometry (B). S2MtEGFP cultures were induced with 700 μM of CuSO₄. At day 5 of induction they were observed in a confocal microscopy and at different times after induction they were evaluated by flow cytometry. For confocal microscopy: (I), optical microscopy; (II), fluorescence microscopy; (III) merged image. For flow cytometry they were observed at days 0, 2, 5, 8, 9 and 12 of induction and the % of fluorescent cells is indicated as M1. Data are representative of independent experiments

Fig. 4.

Kinetics of EGFP intensity expression by S2MtEGFP cells as evaluated by spectrofluorimetry. S2MtEGFP cell culture was induced with 700 μM of CuSO₄ and at different times the EGFP fluorescence emission was detected at 510 nm (A) and the kinetics curve was recorded (B). Data are representative of independent experiments

Improving heterologous protein expression by using sodium butyrate (NaBu).

The effect of NaBu on the EGFP expression by CuSO₄ induced S2MtEGFP cells was analyzed. Figure 5 shows that the NaBu (5 mM) treatment led to a decreased cell growth. At day 4 of induction, cell cultures treated with NaBu attained 0.8 × 10⁷ cells/mL in contrast to control cells that attained 1.6 × 10⁷ cells/mL (Fig. 5A). Following NaBu treatment an increased number of cells expressing the EGFP was observed at 24 h of induction (90%) in comparison to control cell cultures (75.6%) (Fig. 5B). Moreover, the intensity of fluorescence showed by the treated culture sharply increased from 54 units in control cultures to 257 units in the treated ones (Fig. 5C), indicating a remarkable effect of NaBu in promoting the EGFP expression in induced cells. When a higher NaBu concentration (7 mM) was used, the enhancement of EGFP expression was of lower magnitude, possibly due to a higher detrimental effect on the cell metabolism (data not shown).

Fig. 5.

Kinetics of cell growth (A), % of fluorescent cells (B) and fluorescence intensity (C) of S2MtEGFP cells induced with 700 μM of CuSO₄ and treated with 5 mM of NaBu. At day 3, cell cultures were induced with CuSO₄ and/or treated with NaBu. Cell samples were periodically taken for evaluation of viable cell concentration by hematocytometer or fluorescence by flow cytometry. ○, S2MtEGFP cells; •, S2MtEGFP cells + 700 μM of CuSO₄; Δ, S2MtEGFP cells + 5 mM NaBu; ▴, S2MtEGFP cells + 700 μM of CuSO₄ + 5 mM NaBu. Arrows indicate the time of induction at day 3. Results are the average of at least three independent experiments. *: statistically different

Discussion

The control and improvement of transfected gene expression and recombinant protein synthesis is a central point of study for several purposes in biology, engineering and medicine when one consider fundamental research or immunobiological production.

In the present study we have investigated the influence of various parameters for the optimization of high gene expression in stably transfected Drosophila melanogaster S2 cells. The enhanced green fluorescent protein (EGFP) reporter gene, under the control of the inducible metallothionein (Mt) promoter and co-transfected with the hygromycin selection gene was employed in the study. Protocols for vector transfection and gene induction in cell cultures were established.

Several parameters have to be considered for improving gene expression optimization. First of all, together with the gene vector construction, an efficient and reliable transfection procedure is required to provide the recombinant cell population with a high percent of properly transfected cells. For this purpose standardizations in transfection or co-transfection protocols must take into account parameters such as the ratio of vectors (expression and selection), the cell concentration, the metabolic state of the cell population, the concentration of transfection and selection reagents and time and temperature of incubation.

In the present study we have established an optimized protocol for gene co-transfection (Table 1) of S2 cells using 20 μg of cellfectin: 5 × 10⁶ cells seeded and cultured for 24 h in T-25 flasks (5 mL), ration of expression: selection vectors of 20:1 (2 μg DNA) and a transfection incubation period of 5 h at 28 °C in a volume of 500 μL. Hygromycin selection was performed with 600 μg/mL of hygromycin for 2 weeks. The above described transfection protocol led to an enriched S2MtEGFP polyclonal cell population (88% fluorescent cells).

Following the transfection step, it was of importance to establish parameters of optimized gene expression based on the promoter induction. Experiments were carried out to optimize the concentration of CuSO₄ to induce the Mt promoter. Kinetic parameters of cell growth (cell/mL), cell expressing the gene (% of fluorescent cells) and the degree of gene expression intensity (fluorescence intensity) were analyzed by confocal microscopy, flow cytometry and spectrofluorimetry, in suspension cell cultures, mimicking large scale conditions. S2MtEGFP cells were shown to be not influenced, at their exponential growth phase, by the CuSO₄ induction, even though some detrimental effects could be observed after the stationary phase, leading to a higher loss of viability when compared to non-induced cultures. S2MtEGFP cells could, in these conditions, attain a cell concentration of 1.5 to 2 × 10⁷ cells/mL and expressed EGFP as soon as 5 h after CuSO₄ induction. 700 μM of CuSO₄ were able to induce EGFP expression in about 85% of cells with heterogeneous fluorescence intensity, which reached an average of 142 units, after 5 days of induction (Fig. 2–4). Nevertheless, the overall data of cell growth and EGFP expression were substantially affected when CuSO₄ induction was performed at the beginning of cell culture together with cell seeding, indicating a detrimental effect of CuSO₄ on the cell metabolism during the lag phase (data not shown) and the importance of CuSO₄ induction of the EGFP gene during the exponential cell growth phase.

We found a high heterogeneity of EGFP expression among the cells in the population. This could be due to cells harboring plasmids with rearrangements, since plasmid DNA transfection into a cell culture leads to a high frequency of genetic instability (Calos et al. 1983; Razzaque et al. 1983). Besides, since the expression of an heterologous transfected gene, in the cell context, depends on its properly insertion into the cell genome, it is conceivable that a variable number of gene copies, upon cell transfection and gene integration, became inserted in not transcribed regions of the genome. So, even efficient procedures of gene transfection may lead to gene integrated copies that are not expressed.

Based on published studies (Dorner et al. 1989; Chen et al. 2002; Li and Li 2006; Zhao et al. 2006; Ping et al. 2006; Song et al. 2006), sodium butyrate (NaBu), is a histone deacetylase inhibitor, affecting the chromatin structure and leading to the destabilization of nucleosomal structure. As a consequence, the NaBU influence on chromatin exposure may facilitate binding of transcription factors to DNA resulting in an increased and deregulated cell protein synthesis.

We have investigated the enhancement of EGFP expression by S2MtEGFP cell treatment with NaBu. As expected, the cell treatment with NaBu led to loss of cell multiplication and viability, since the NaBu is a potent inducer of G1 cell cycle arrest and provokes apoptosis by activating an intrinsic apoptotic pathway (Li and Li 2006). On the other hand NaBu treatment may expose cell genome regions bearing an heterologous transfected gene and so foster the synthesis of their products (Dorner et al. 1989; Ping et al. 2006; Song et al. 2006). Indeed, our data clearly show that S2MtEGFP cell treatment with NaBu led to a decreased cell multiplication (from 1.6 × 10⁷ in controls with only addition of CuSO₄, to 0.8 × 10⁷ cells/mL) and decreased viability, which was accompanied by not only a clear cut enhancement of fluorescence intensity of the cell population (∼5 fold) but also an early increase in the percent of fluorescent cells among the induced cell population (from 75.6% in controls to 90%) at one day post-induction (Fig. 5).

Drosophila S2 cells is a promising cell system for heterologous gene expression due to its characteristics of easy handle cell culture procedures associated to the possibilities of stable and induced gene expression. EGFP, as a reporter gene, is a useful tool to establish suitable parameters for improving heterologous protein expression. We show here in this study an optimized vector transfection protocol that enabled us to establish a primary stable transfected S2MtEGFP cell population that once induced by CuSO₄, expressed the recombinant EGFP in a high percentage of the cells. Its expression, by the CuSO₄ induced cell population, could be further sharply enhanced by sodium butyrate (NaBu). The procedure shown here was developed in view of a general application for gene expression in the stable transfected Drosophila S2 cell system.

Abbreviations

EGFP

Enhanced green fluorescent protein gene

pMt

Metallothionein promoter

S2

Schneider 2 Drosophila melanogaster cells

S2MtEGFP

S2 cells transfected with Mt inducible plasmid containing the EGFP gene

NaBu

Sodium butyrate

CuSO₄

Copper sulfate

DES

Drosophila expression system

Hygro

Hygromycin