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An improved chloramphenicol acetyltransferase assay for Plasmodium falciparum transfection
Molecular & Biochemical Parasitology 136 (2004) 287–296
An improved chloramphenicol acetyltransferase assay for Plasmodium falciparum transfection Stuart J. Lucas∗,1 , Anthony A. Holder Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK Received 17 March 2004; accepted 19 March 2004 Available online 25 May 2004
Abstract Chloramphenicol acetyltransferase (CAT) is a popular choice as a reporter gene in transgenic studies in many different organisms, including Plasmodium falciparum. For experimental investigations into transfection efficiency and gene expression a robustly quantitative assay is of great value. On investigation the published protocol for CAT assay of P. falciparum was found to be prone to saturation due to the long incubation time; moreover, cellular material extracted from the parasite increased the enzyme activity. A new protocol was developed which is quantitative across a range of three orders of magnitude of CAT activity, takes account of the cellular extract effect, and is more rapid than the established method. The value of these improvements was demonstrated by analysing the effects of parasitaemia and amount of plasmid on transfection efficiency with both old and new methods. © 2004 Elsevier B.V. All rights reserved. Keywords: CAT assay; Chloramphenicol acetyltransferase; Quantitative assay; Reporter gene; Transient transfection; Malaria; Plasmodium falciparum
1. Introduction Plasmodium falciparum causes the most virulent form of human malaria. It is responsible for the majority of the 2–3 million malaria deaths estimated to occur annually, making it a research priority. The development of genetic manipulation techniques for this clinically important parasite over the last few years is opening up many avenues for identifying and characterising potential drug and vaccine targets (reviewed in [1]). Heterologous reporter genes that can be introduced and then readily detected facilitate the molecular genetic analysis of any organism. Such genes are particularly useful for probing the mechanisms of gene expression and regulation. The chloramphenicol 3-O-acetyltransferase (CAT, EC 2.3.1.28) gene has become a reporter gene of choice for studying gene expression in many kinds of eukaryotic cells, Abbreviations: BSA, bovine serum albumin; CAT, chloramphenicol 3-O-acetyltransferase; LSC, liquid scintillation counting; PcDT, Plasmodium chabaudi dihydrofolate reductase/thymidylate synthase; TLC, thin layer chromatography ∗ Corresponding author. Tel.: +44 20 7594 3979; fax: +44 20 7594 3973. E-mail address: stu [email protected] (S.J. Lucas). 1 Present address: Imperial College Department of Virology, WrightFleming Institute, Norfolk Place, London, W2 1PG, UK. 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.03.017
following its first use by Gorman et al. [2]. The CAT enzyme is a selectable marker for the broad-spectrum antibiotic chloramphenicol, and there are highly sensitive assays of its activity available, using radioactive or chromogenic variants of its substrates, chloramphenicol and acetyl-CoA. Moreover there is no analogous enzyme in most eukaryotic cells, and so no background activity to take into account. CAT has been used from the very beginning of the genetic manipulation of P. falciparum [3]. While the gene has not been used as an effective selectable marker in this organism, it has been used as a reporter to demonstrate successful transfection [3], compare transfection efficiencies [4] and assess promoter activities [5,6]. However, in these earlier reports no evidence was presented to show that a robustly quantitative CAT assay was used. Enzyme assays are quantitative if the amount of product formed increases linearly with enzyme concentration; this usually requires substrates to be present in excess throughout the assay period. The assay conditions described by [3] had acetyl-CoA in excess, but the radioactive substrate, [14 C]chloramphenicol, was at 0.126 M—a concentration lower than the Km of CAT for chloramphenicol, which has been measured at 6–7 M [7]. Under these conditions it is unlikely that the amount of product formed will vary linearly with enzyme concentration, particularly in an overnight incubation at 37 ◦ C. Over a long period sub-
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strate concentration may drop significantly, the product may degrade, and the enzyme may lose activity. Previously, P. falciparum CAT products were analysed by thin layer chromatography (TLC) following extraction with ethyl acetate [3]. The different acetylated forms separated from unacetylated chloramphenicol in this way must then be quantified using a phosphorimager to measure the relative intensities of the radioactivity corresponding to each chemical species. This is a labour-intensive procedure. Phase separation with different organic solvents has been shown to be more efficient than ethyl acetate in separating products and substrate [7]. This procedure eliminates the chromatography step and the amount of product can be measured rapidly by liquid scintillation counting (LSC). A quantitative CAT assay would be a valuable tool for accurately measuring gene expression, for example in identifying promoter sequences or studying mechanisms that regulate expression levels. Therefore the assay protocol described previously was tested to find out whether or not it was quantitative, and whether it could be improved. In the course of these experiments it was observed that cellular extracts from P. falciparum stabilise CAT activity. An improved CAT assay was developed for P. falciparum, which is both quantitative and less time-consuming than that previously described. 2. Materials and methods 2.1. Parasites and culture Clone 3D7 parasites, originally obtained from D. Walliker, were used throughout. The culture method was modified from that of Trager and Jensen [8]. P. falciparum blood stages were maintained in human erythrocytes at up to 15% parasitaemia and 0.5–1% haematocrit in RPMI 1640 enriched with 24 mM NaHCO3 , 25 mM HEPES, 9 mM glucose, 25 g ml−1 gentamycin, 16 g ml−1 hypoxanthine, 5% (w/v) Albumax I and 0.1% (w/v) l-glutamine added fresh before use. Cultures were gassed with a mixture of 7% CO2 , 5% O2 and 88% N2 and incubated at 37 ◦ C. Parasites were synchronised using the Percoll density gradient method [9]. 2.2. Transient transfection of P. falciparum All transfections used the plasmid pHC1-CAT [4], which was a gift from A. Cowman, and was prepared using the QiaFilter Maxiprep kit (Qiagen). Transient transfection was carried out by a modification of the method of Wu et al. [3]. Prior to transfection 50 g plasmid was diluted into 200 l incomplete cytomix (120 mM KCl, 5 mM MgCl2 , 0.15 mM CaCl2 , 2 mM EGTA, 25 mM HEPES in 10 mM potassium phosphate buffer, pH 7.6), which was then mixed gently with an equal volume of erythrocytes containing early ring stage parasites. Except where otherwise stated, the mixture was electroporated in a 0.2 cm cuvette at 300 V, 950 F using
a Bio-Rad Gene Pulser II and transferred immediately to 15 ml pre-warmed medium. All cultures were then gassed and incubated at 37 ◦ C. 2.3. Extracting cellular material for CAT assay Transfectants were cultured for 40 h, by which point the majority of parasites were segmenting schizonts, and then harvested as described by [3]. Parasitized erythrocytes were first washed with phosphate-buffered saline (PBS) by centrifugation, and then resuspended in two pellet volumes of PBS containing 0.15% (w/v) saponin, to disrupt the erythrocyte plasma membrane. After 10 min at 37 ◦ C the cells were spun down again and the haemoglobin-containing supernatant removed. Parasite pellets were stored at −80 ◦ C if necessary. After thawing the cells were resuspended in 1 ml of TEN buffer (40 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, pH 7.6), spun down, and resuspended again in 120 l 0.25 M Tris–HCl, pH 7.6. Next the samples were subjected to three cycles of fast freeze-thawing by transferring them between dry ice and a 37 ◦ C water bath. After a final incubation at 65 ◦ C for 10 min (to inactivate deacetylases) the extracts were centrifuged at top speed for 2 min in a microcentrifuge, and the supernatant containing the CAT enzyme removed. 2.4. Measuring CAT activity The basic assay was modified from the method described by [3]. To a sample of P. falciparum cell extract was added 5 l of a 10 mg ml−1 acetyl-CoA solution (final concentration 500 M), and 0.25 M Tris–HCl, pH 7.6 to a volume of 123 l. Reactions were started by adding 2 l (50 nCi) 14 C-labelled chloramphenicol (Amersham Biosciences No. CFA754), and incubated at 37 ◦ C for a specified time. Purified CAT was purchased from Promega. For analysis by TLC, CAT assay products were first extracted by phase separation, adding 400 l ethyl acetate, vortex mixing for 30 s, and then spinning in a microcentrifuge at top speed for 2 min. The organic (upper) phases were transferred into new tubes, and the ethyl acetate evaporated off in a Savant Speed Vac Concentrator (Model RH 40-11). Once dry, the products were redissolved in 10 l ethyl acetate and spotted onto silica coated aluminium TLC plates (Whatman No. 4420-221), allowing spots to dry between each application. The plates were then placed in a glass chromatography tank with a 19:1 (v/v) mixture of chloroform: methanol mobile phase. After 45 min to 1 h plates were removed, dried, and placed overnight in a phosphorimager cassette. The following day the cassette was scanned using a Molecular Dynamics Storm 860 phosphorimager, the data being analysed with ImageQuant 5.0 software. For LSC analysis, CAT assay products were extracted by phase separation, adding 300 l mixed xylenes, vortex mixing for 30 s, and spinning in a microcentrifuge at top speed for 3 min at 4 ◦ C. The organic (upper) phases were trans-
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ferred to fresh tubes and back-extracted by adding 100 l 0.25 M Tris–HCl, pH 7.6, vortex mixing and centrifugation as before. The back extraction was repeated and then the organic phases were transferred to scintillation vials with 2 ml Ready-Safe scintillant (Beckman). The activity of the samples was then measured on a Beckman LS6000IC scintillation counter, with a program set to count 14 C for 1 min per sample. 2.5. Estimation of total protein concentration Protein concentration was estimated using the Bio-Rad Protein Assay, using a 96-well plate, microassay format as recommended by the manufacturers. Bovine serum albumin was used as a standard protein and absorbance readings were measured using a Dynex MRX-TC 2 plate reader.
3. Results 3.1. Liquid scintillation counting is a convenient alternative to thin layer chromatography A LSC protocol was adapted for use with P. falciparum, and compared with the established TLC method. A set of parasites was transfected with pHC1-CAT under varying electroporation conditions, grown for 44 h, harvested as described and assayed for CAT activity. Half of the CAT reaction products were extracted with ethyl acetate and separated by TLC, and the other half by extraction with mixed xylenes. Products were measured by phosphorimager analysis and LSC respectively. Both sets of data were plotted as a percentage of the product from a positive control reaction containing 1 U commercially purified CAT (Fig. 1A). All the samples tested gave very similar results and variability for both methods, demonstrating that they are interchangeable. The LSC data were a consistently lower percentage of the control (average 5.8 ± 3.4%; P < 0.001 by paired t-test) for all samples; in most situations this difference would have no importance, but it suggests that at high CAT activities phosphorimager detection may saturate slightly sooner than LSC. To assess the efficiency of xylene extraction in separating products from substrate, controls of the CAT assay mix without enzyme were examined (Fig. 1B). In the initial xylene extraction less than 5% of the total radioactivity partitioned into the organic phase, and after two back extractions this was reduced to below 0.1%. At this level the radioactivity was indistinguishable from the background with scintillation cocktail alone, showing that essentially no substrate was extracted with the acetylated products. 3.2. Long incubations lead to assay saturation A curve was plotted of the amount of product using purified CAT in the previously reported assay protocol, to assess
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the method’s suitability for quantitative studies. Purified enzyme was diluted to 1 U l−1 in assay buffer, and two serial 100-fold dilutions were made from this stock. From these, assays were set up using 10 different quantities of enzyme covering four orders of magnitude: 2, 1, 0.4, 0.1, 0.04, 0.01, 0.004, 0.001, 0.0004 and 0.0001 U enzyme in a 125 l reaction. Each assay was set up in triplicate and incubated overnight at 37 ◦ C. The following day, the products from half of each reaction were measured by liquid scintillation counting. As shown in Fig. 2, the resulting curve was not linear. For enzyme activities below 0.001 U the radioactivity of the products was at background levels, while at higher enzyme activities saturation was observed in the assay. The only part of the curve that approximated to linearity was from 0.001 to 0.01 U (Fig. 2C), which is an extremely restricted linear range. The assay was saturating probably because one or both of the substrates was being used up. Each reaction contained about 100 000 counts per minute (cpm) (Fig. 1B) and with 2 U of CAT nearly 50% of the chloramphenicol had been acetylated by the end of the assay (Fig. 2, data for half the reaction). As noted in the introduction, chloramphenicol was not present in excess, so the substantial drop in substrate concentration at high CAT activities was bound to result in reduced product. 3.3. Effect of cell extract In preliminary experiments to improve the CAT assay, purified enzyme gave maximum acetylated product after 1–2 h incubation, and the amount of product dropped if incubated overnight. In contrast, the acetylated chloramphenicol produced when CAT-containing extracts from transfected parasites were used in CAT assays continued to increase when the extracts were incubated overnight (data not shown). This suggested that something in the P. falciparum extract stabilises the enzyme or its product. To test this hypothesis, P. falciparum cell extract was prepared from untransfected parasitized erythrocytes by the method used to obtain CAT, and verified to have no detectable CAT activity. Assays were then set up with 0.1 U purified CAT and 0, 50 or 100 l cell extract, the last sample imitating the conditions when CAT was extracted from P. falciparum. As shown in Fig. 3A, saturation occurred by 1 h, with no further increase in products by 2 h. However, after overnight incubation the amount of product dropped significantly, unless cell extract was present. A similar effect was observed in a 2-h time course with 0.01 U enzyme (Fig. 3B). In the presence of 100 l cell extract the amount of product increased linearly with time, whereas without cell extract the activity flattened out after about 1 h and then dropped on continued incubation. A reduced amount of cell extract (10 l per 125 l reaction) had an intermediate effect: the activity climbed throughout the 2-h time course, but the rate dropped with time.
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Fig. 1. (A) Ring stage parasites were transfected under a variety of conditions, harvested, and CAT assay reactions set up as described. After overnight incubation, the reaction mixtures were each split in half. The acetylated products from one half were separated and quantified using TLC and phosphorimaging (grey bars), and from the other half by xylene extraction and LSC (white bars). Results are expressed as a percentage of the control reactions, which contained 1 U purified CAT. Transfection conditions were: (a) 2000 V, 25 mF; (b) 280 V, 960 mF; (c) 250 V, 960 mF; (d) 300 V, 960 mF. Error bars show standard deviation of triplicate samples. (B) In all samples 50 nCi of d-threo-[dichloroacetyl-1-14C]chloramphenicol were diluted to a final volume of 125 l in 0.25 M Tris–HCl buffer, pH 7.6. Activity was measured after no extraction (Total counts), extraction with mixed xylenes (Extracted), one back extraction of the mixed xylenes using 100 l 0.25 M Tris–HCl buffer (Extr. + Back Extracted) or two such back extractions (Extr. + 2× Back Extracted). Error bars show standard deviation of duplicates.
The decrease in measured activity after extended incubation shows that neither the enzyme activity nor the products are entirely stable at 37 ◦ C. Product breakdown alone would produce an equilibrium when the rate of breakdown matched the rate of product formation, causing the measured activity to reach a plateau over time, but not to decrease until the substrate is exhausted. Under the conditions used in Fig. 3B, substrate was still abundant by the end of the experiment. Similarly, if the enzyme was deteriorating but the products were stable, the measured activity would reach a plateau but not decrease.
P. falciparum cell extract stabilised both enzyme activity and products. The amount required to give maximum CAT activity was measured by including different amounts of cell extract in assays of 0.01 U CAT incubated for 65 min. The effect of parasite cell extract reached a maximum at a concentration of 84 g of protein per ml, with a further 3-fold increase in concentration conferring no additional gain in activity (Fig. 4). Parallel assays were set up using the same concentration of bovine serum albumin (BSA). Including BSA in the assay also increased the amount of product in a dose-dependent fashion, but even at the highest
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Fig. 2. A wide range of enzyme activities were obtained by diluting commercially purified CAT and adding them to a standard CAT assay mixture. Reactions were incubated overnight at 37 ◦ C, and the acetylated chloramphenicol produced measured by xylene extraction and liquid scintillation counting. Error bars are standard deviation of triplicate samples: (A) entire data range; (B) expansion of shaded area of (A); (C) expansion of shaded area of (B). The points in the linear region of the curve are picked out with filled diamonds.
concentration tested the yield did not approach the maximum activity seen with cell extract. All subsequent reactions using purified CAT were supplemented with P. falciparum cell extract at a final concentration of 84 g of protein per ml. 3.4. Developing a quantitative protocol for CAT assay Long incubations may lead to product saturation as the chloramphenicol substrate is used up, and loss of product occurs—although this loss is ablated in the presence of cell extract. To solve these problems we measured the initial rate of the reaction, rather than the amount of product after a certain incubation period. This method is widely used in enzyme kinetics because it avoids the problems of enzyme instability and product saturation; in practice, the initial rate is often taken as the reaction rate before 10% of the substrate has been used up. A wide range of CAT concentrations was freshly made in triplicate in an assay mix supplemented with P. falciparum
cell extract. A time course at each concentration was carried out, with typical data shown in Fig. 5A. The initial rate of reaction for each curve was calculated by linear regression analysis of all the points in the linear section of the curve. These rates, in cpm min−1 , were plotted against CAT activity in U (Fig. 5B). Owing to the broad range of data points, the values were plotted on a logarithmic scale. The relationship between initial rate and enzyme concentration would be predicted to be a straight line of the form: Vi = M[E]T Where [E]T is the total enzyme concentration, Vi is the measured initial rate of reaction, and M is the gradient of the graph in Fig. 5B. M was calculated separately for the values at each data point and the mean of these values was 12 193 ± 2142 cpm min−1 U−1 . The line of best fit computed from this value is plotted on Fig. 5B, with the faint lines either side indicating the standard deviation.
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Fig. 3. CAT assays were set up using known amounts of purified CAT in the presence of varying amounts of P. falciparum cell extract as indicated. Total volume of all assay reactions was 125 l. (A) Reactions contained 0.1 U commercially purified CAT and incubated at 37 ◦ C for 1, 2 or 16 h before stopping the reaction by xylene extraction of the products. Error bars show the standard deviation of duplicates. (B) Time course of reaction for 0.01 U purified CAT. At each time point reactions were stopped by xylene extraction. Error bars show the standard deviation of duplicates. The line of best fit to the ‘100 l cell extract’ dataset was calculated by linear regression analysis and is plotted on the graph.
3.5. Comparison of old and new CAT assay protocols in the transient transfection assay The new, quantitative protocol was compared with the previously published method firstly by studying the effect of initial parasitaemia on transfection efficiency in a transient transfection assay. Starting with a P. falciparum culture containing early ring stages at about 12% parasitaemia, aliquots were mixed with unparasitized erythrocytes to give a range of parasitaemias, which were measured by counting thin blood smears. At each parasitaemia, four aliquots of cells were electroporated with 50 g pHC1-CAT, cultured for 40 h, harvested, and assayed for CAT activity. Two aliquots were then assayed using the TLC-based published method (Fig. 6A), and two using the new LSC-based protocol (Fig. 6B). To apply the new protocol, the cell extract from each transfected cell culture was used to prepare three 30 l aliquots for CAT assay. The total protein concentra-
tion of each extract was estimated using the BCA assay and each sample was supplemented with cell extract from untransfected parasites to the same total protein concentration, as required. The three assay mixtures from each extract were then incubated for three different times, stopping the reaction each time by xylene extraction of the products. The amount of product at the three time points was used to calculate the initial rate of reaction, and these rates were compared with the standard curve (Fig. 5B) to give a measure of the total CAT activity at each parasitaemia (Fig. 6B). The differences between the two datasets are immediately apparent. Using the published assay method, it appears that the relationship between parasitaemia and CAT activity is described by a saturation curve across the whole range. However, the quantitative protocol shows that activity actually increases linearly across the range until at least 12% parasitaemia; previous observations (data not shown) found that at higher densities the parasites become stressed and
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Fig. 4. P. falciparum cell extract obtained from 2 × 109 erythrocytes infected by schizonts at 10% parasitaemia was found to contain 350 mg ml−1 total protein. Bovine serum albumin (Sigma) was diluted to the same concentration in 0.25 M Tris–HCl, pH 7.6. 0.01 U commercially purified CAT enzyme was freshly diluted into reaction aliquots of 125 l, which included 0, 10, 30, or 90 l P. falciparum cell extract or BSA as indicated. All reactions were incubated for 65 min at 37 ◦ C, and then stopped by xylene extraction followed by liquid scintillation counting of the acetylated products. Error bars show the standard deviation of triplicate samples.
some die under the culture conditions used. The quantitative method also gives a measure of CAT activity, which the old protocol cannot. A similar, although less marked effect, was observed when the amount of plasmid used in the transfection was varied (Fig. 7). In this case, rings at 2% parasitaemia were used to minimise the saturation due to parasitaemia observed in Fig. 6A. Again the data obtained by the published method suggests that when more than 100 g plasmid is used, transfection efficiency rapidly saturates. However, the quantitative protocol suggests that while efficiency does begin to saturate at this high plasmid concentration, the effect is much less dramatic. Additionally, this experiment allows the lower detection limit of the two methods to be compared; neither method detected activity significantly above background when 2 g plasmid was used, but the quantitative protocol detected activity from 5 to 10 g plasmid where the published method did not.
4. Discussion The experiments reported here demonstrate that the CAT assay previously described for use with P. falciparum is qualitative, but not quantitative; that is, it can show that under the same conditions sample A contains more CAT than sample B, but not how much. Several refinements were made to the assay, which should be valuable for the quantitative analysis of gene expression and regulation in P. falciparum. At the final stage of the assay, separation and measurement of CAT reaction products was streamlined by using xylene extraction and LSC instead of TLC and phosphorimaging. This technique is at least as accurate, repro-
ducible and sensitive as the established method, and is less laborious. During the development of this assay it was observed that purified CAT enzyme is stabilised by the addition of P. falciparum cell extracts during prolonged incubation at 37 ◦ C. Similarly, Mandel [10] observed that purified CAT subjected to the 65 ◦ C preheating step to inactivate cell-derived deacetylases, lost at least 50% of its activity. Therefore if purified CAT is used as a standard for quantitating assay data it must be stabilised in some way. BSA is often added to enzyme reactions to increase stability, but in this study cell extract from untransfected P. falciparum was significantly more effective and it replicates more closely the conditions in the samples under test. The assay products are also stabilised by the presence of P. falciparum cell extract. It has previously been reported that cellular material from Saccharomyces cerevisiae increases activity in the CAT assay in a concentration dependent manner, both for endogenously produced and for purified bacterial enzyme [11]. This study also found that desalting by gel filtration greatly reduced the effect of the cell extract, implicating small molecules as being largely responsible. From assays using known amounts of CAT it was found that the established assay is linear only across a small range of enzyme concentrations. At high CAT activities the long incubation time leads to saturation of the assay by chloramphenicol depletion. Levels of CAT sufficient to cause this problem were generated in transient transfection experiments, as shown in Fig. 6A. Saturation of the assay makes it misleading; for example from Fig. 6A one might conclude that the difference in transfection efficiency at 5 or 10% parasitaemia is <20%. However, Fig. 6B shows that the difference is much greater than this. Similarly Fig. 7 shows
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Fig. 5. Initial rates of reaction were measured for a range of quantities of purified CAT enzyme and used to generate a standard curve. (A) Typical data showing time courses for 0.01, 0.003 and 0.001 U CAT as indicated. All reactions contained 30 l P. falciparum cell extract in a total volume of 125 l. Error bars show standard deviation of triplicate samples. Lines of best fit were calculated by linear regression analysis of all points lying within the initial linear phase, and plotted on the graph. (B) The gradients of the lines of best fit were taken as measures of the initial reaction rate and plotted against enzyme activity, with error bars showing the standard error of these values. At each point on this graph, y/x was calculated to obtain an estimate of the gradient of the standard curve. The mean of these estimates was found and used to plot a line of best fit to the data (solid line). One standard deviation of this line of best fit is plotted above and below the line itself (dashed lines).
that using plasmid in higher quantities than normally used in transient transfections (typically 50 or 100 g [3–5]) gives a greater increase in transfection efficiency than indicated by the published method. These differences could be important when, for example, trying to boost detection of activity from a weak promoter. In the latter example the new protocol also detected smaller amounts of CAT than the published method. A likely reason for this is that when a very small amount of CAT has been produced, an overnight incubation is long enough for it to degrade. We would expect the stabilising effect of P. falciparum cell extract to solve this problem, but in this instance using a lower parasitaemia (2% rather than 10%, to avoid the saturation seen for the old protocol in Fig 6A) and dividing the extract in two (half for each assay method) meant that the cell extract in the samples measured by the published method would have been one tenth of its normal
amount, which, as shown by the middle dataset in Fig. 3B, gives only partial stabilisation of the assay. It is expected that a higher parasitaemia would give improved detection of the lowest amounts of plasmid tested but greater saturation of the assay when using higher amounts. The results shown here contrast with those of Wu et al. [3], who detected CAT activity following transfection with as little as 1 g of plasmid. In that study the starting parasitaemia was 10%, not 2%, and CAT expression was driven by the hsp86 5 upstream region, which is believed to be stronger than the Calmodulin promoter used in pHC1-CAT. A fully quantitative assay protocol was developed for the reporter enzyme CAT, following transfection of the gene into P. falciparum. This assay will be of considerable value as a positive control for transfection, in comparing transfection efficiencies, and in characterising promoters. These authors have since used the improved assay to study the activities
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Fig. 6. Effect of parasitaemia on transfection efficiency measured by old and new protocols. (A) Cell extracts were prepared from duplicate cultures of transiently transfected erythrocytes as described in Section 2. One-hundred microliters of each extract were included in CAT assay reactions which were incubated overnight before xylene extraction and liquid scintillation counting. The chart shown includes data from two different experiments carried out on different days on different parasite cultures. Error bars show the standard deviation of between two and four samples. (B) Cell extracts from duplicate cultures were assayed for total protein concentration. CAT assay reactions were then set up containing 30 l extract from one of the transfected cultures, supplemented with untransfected P. falciparum cell extract to give a total protein concentration equal to that of the 12% parasitaemia cultures. For each culture three assays were set up which were incubated for 0, 1 or 2 h before extraction with mixed xylenes and liquid scintillation counting. Linear regression analysis was used to calculate the initial rate of reaction for each culture, which was compared with the standard curve in Fig. 5B to obtain a measure of the CAT activity present (plotted above). Error bars show the standard deviation of the values for initial rate estimated at each time point. A line of best fit was plotted by linear regression.
Fig. 7. Effect of amount of plasmid used on transfection efficiency measured by old and new protocols. A range of quantities of pHC1-CAT from 2 to 200 g were diluted into 200 l incomplete cytomix and transfected into 200 l aliquots of packed rbcs parasitized with 2% rings as described in the methods section. On extracting cellular material for CAT assay, each extract was divided in half, one half of the extract being assayed by the old protocol and one by the new protocol. Activities from the old protocol (䉫) were measured by liquid scintillation counting and are plotted against the left-hand axis. Samples from the new protocol were incubated for 0, 135 and 270 min before extraction and scintillation counting. Linear regression analysis was then used to estimate the initial rate of reaction and compare this with the standard curve in Fig. 5B to give values (䊏) that are plotted against the right-hand axis. Error bars show the standard deviation of duplicate or triplicate transfections.
of a range of promoters (manuscript in preparation). The new protocol is somewhat more labour intensive than the published one, as it requires at least three time points to be measured for each sample under test. However, this requirement is balanced by the shorter incubation time and simpler
separation and quantification of products, so that the entire assay can be completed in 1 day rather than 3 days. Furthermore, it gives an absolute measure of the CAT activity in a sample, rather than just a relative one; e.g. after transfection at 10% parasitaemia, 0.025 U CAT are generated from
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the calmodulin/PcDT promoter in pHC1-CAT. If this measure were combined with an accurate measure of transfection efficiency, the amount of CAT produced per transfected parasite could be calculated. Another reporter gene widely used in Plasmodium transfection studies is firefly luciferase, which can be quantitated by producing a standard curve in parallel with each experiment [12]. More recently a second luciferase, from Renilla regiformis, has also been applied to P. falciparum [13]. The luciferase assay is quicker than the CAT assay, although the simplification of the detection method described here reduces the difference. In some situations the CAT assay may be preferable as the luciferase gene product is relatively short-lived. For example, in a time course experiment luciferase produced from a promoter with a fairly small expression window might decay before the next measurement. To date the relative sensitivities of these two reporter genes have not been directly compared in P. falciparum. Whatever the relative advantages and disadvantages of each, having both of them available opens up a range of experiments that are not possible with one alone, such as dual labelling by fusing them to two different proteins, or using one to measure expression while using the other as an internal control. The observations made in this study illustrate the importance of thoroughly characterising an assay system before applying it to biological questions. Further improvements to this and other assays, as well as new alternatives such as commercially available fluorimetric and ELISA-based CAT assays, should be investigated, as they would be of significant benefit to the field.
Acknowledgements Thanks to M. Grainger for providing parasites and assistance with parasite culture, to A. Cowman for the gift of the pHC1-CAT plasmid, and to M. Hensmann for useful discussion. S.J.L. was in receipt of a MRC Studentship.
References [1] Waterkeyn JG, Crabb BS, Cowman AF. Transfection of the human malaria parasite Plasmodium falciparum. Int J Parasitol 1999;29(6):945–55. [2] Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 1982;2(9):1044–51. [3] Wu Y, Sifri CD, Lei HH, Su XZ, Wellems TE. Transfection of Plasmodium falciparum within human red blood cells. Proc Natl Acad Sci: USA 1995;92(4):973–7. [4] Crabb BS, Triglia T, Waterkeyn JG, Cowman AF. Stable transgene expression in Plasmodium falciparum. Mol Biochem Parasitol 1997;90(1):131–44. [5] Crabb BS, Cowman AF. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum. Proc Natl Acad Sci: USA 1996;93(14): 7289–94. [6] Wickham ME, Thompson JK, Cowman AF. Characterisation of the merozoite surface protein-2 promoter using stable and transient transfection in Plasmodium falciparum. Mol Biochem Parasitol 2003;129(2):147–56. [7] Sankaran L. A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail. Anal Biochem 1992;200(1):180–6. [8] Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193(4254):673–5. [9] Freeman RR, Holder AA. Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000 mol wt form expressed on the surface of Plasmodium falciparum merozoites. J Exp Med 1983;158(5):1647–53. [10] Mandel HG. Thermal instability of chloramphenicol acetyltransferase: assay revisions required. Anal Biochem 1995;230(1): 191–3. [11] Alipour H, Eriksson P, Norbeck J, Blomberg A. Quantitative aspects of the use of bacterial chloramphenicol acetyltransferase as a reporter system in the yeast Saccharomyces cerevisiae. Anal Biochem 1999;270(1):153–8. [12] Goonewardene R, Daily J, Kaslow D, et al. Transfection of the malaria parasite and expression of firefly luciferase. Proc Natl Acad Sci: USA 1993;90(11):5234–6. [13] Militello KT, Wirth DF. A new reporter gene for transient transfection of Plasmodium falciparum. Parasitol Res 2003;89(2):154–7.
An improved chloramphenicol acetyltransferase assay for Plasmodium falciparum transfection Stuart J. Lucas∗,1 , Anthony A. Holder Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK Received 17 March 2004; accepted 19 March 2004 Available online 25 May 2004
Abstract Chloramphenicol acetyltransferase (CAT) is a popular choice as a reporter gene in transgenic studies in many different organisms, including Plasmodium falciparum. For experimental investigations into transfection efficiency and gene expression a robustly quantitative assay is of great value. On investigation the published protocol for CAT assay of P. falciparum was found to be prone to saturation due to the long incubation time; moreover, cellular material extracted from the parasite increased the enzyme activity. A new protocol was developed which is quantitative across a range of three orders of magnitude of CAT activity, takes account of the cellular extract effect, and is more rapid than the established method. The value of these improvements was demonstrated by analysing the effects of parasitaemia and amount of plasmid on transfection efficiency with both old and new methods. © 2004 Elsevier B.V. All rights reserved. Keywords: CAT assay; Chloramphenicol acetyltransferase; Quantitative assay; Reporter gene; Transient transfection; Malaria; Plasmodium falciparum
1. Introduction Plasmodium falciparum causes the most virulent form of human malaria. It is responsible for the majority of the 2–3 million malaria deaths estimated to occur annually, making it a research priority. The development of genetic manipulation techniques for this clinically important parasite over the last few years is opening up many avenues for identifying and characterising potential drug and vaccine targets (reviewed in [1]). Heterologous reporter genes that can be introduced and then readily detected facilitate the molecular genetic analysis of any organism. Such genes are particularly useful for probing the mechanisms of gene expression and regulation. The chloramphenicol 3-O-acetyltransferase (CAT, EC 2.3.1.28) gene has become a reporter gene of choice for studying gene expression in many kinds of eukaryotic cells, Abbreviations: BSA, bovine serum albumin; CAT, chloramphenicol 3-O-acetyltransferase; LSC, liquid scintillation counting; PcDT, Plasmodium chabaudi dihydrofolate reductase/thymidylate synthase; TLC, thin layer chromatography ∗ Corresponding author. Tel.: +44 20 7594 3979; fax: +44 20 7594 3973. E-mail address: stu [email protected] (S.J. Lucas). 1 Present address: Imperial College Department of Virology, WrightFleming Institute, Norfolk Place, London, W2 1PG, UK. 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.03.017
following its first use by Gorman et al. [2]. The CAT enzyme is a selectable marker for the broad-spectrum antibiotic chloramphenicol, and there are highly sensitive assays of its activity available, using radioactive or chromogenic variants of its substrates, chloramphenicol and acetyl-CoA. Moreover there is no analogous enzyme in most eukaryotic cells, and so no background activity to take into account. CAT has been used from the very beginning of the genetic manipulation of P. falciparum [3]. While the gene has not been used as an effective selectable marker in this organism, it has been used as a reporter to demonstrate successful transfection [3], compare transfection efficiencies [4] and assess promoter activities [5,6]. However, in these earlier reports no evidence was presented to show that a robustly quantitative CAT assay was used. Enzyme assays are quantitative if the amount of product formed increases linearly with enzyme concentration; this usually requires substrates to be present in excess throughout the assay period. The assay conditions described by [3] had acetyl-CoA in excess, but the radioactive substrate, [14 C]chloramphenicol, was at 0.126 M—a concentration lower than the Km of CAT for chloramphenicol, which has been measured at 6–7 M [7]. Under these conditions it is unlikely that the amount of product formed will vary linearly with enzyme concentration, particularly in an overnight incubation at 37 ◦ C. Over a long period sub-
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strate concentration may drop significantly, the product may degrade, and the enzyme may lose activity. Previously, P. falciparum CAT products were analysed by thin layer chromatography (TLC) following extraction with ethyl acetate [3]. The different acetylated forms separated from unacetylated chloramphenicol in this way must then be quantified using a phosphorimager to measure the relative intensities of the radioactivity corresponding to each chemical species. This is a labour-intensive procedure. Phase separation with different organic solvents has been shown to be more efficient than ethyl acetate in separating products and substrate [7]. This procedure eliminates the chromatography step and the amount of product can be measured rapidly by liquid scintillation counting (LSC). A quantitative CAT assay would be a valuable tool for accurately measuring gene expression, for example in identifying promoter sequences or studying mechanisms that regulate expression levels. Therefore the assay protocol described previously was tested to find out whether or not it was quantitative, and whether it could be improved. In the course of these experiments it was observed that cellular extracts from P. falciparum stabilise CAT activity. An improved CAT assay was developed for P. falciparum, which is both quantitative and less time-consuming than that previously described. 2. Materials and methods 2.1. Parasites and culture Clone 3D7 parasites, originally obtained from D. Walliker, were used throughout. The culture method was modified from that of Trager and Jensen [8]. P. falciparum blood stages were maintained in human erythrocytes at up to 15% parasitaemia and 0.5–1% haematocrit in RPMI 1640 enriched with 24 mM NaHCO3 , 25 mM HEPES, 9 mM glucose, 25 g ml−1 gentamycin, 16 g ml−1 hypoxanthine, 5% (w/v) Albumax I and 0.1% (w/v) l-glutamine added fresh before use. Cultures were gassed with a mixture of 7% CO2 , 5% O2 and 88% N2 and incubated at 37 ◦ C. Parasites were synchronised using the Percoll density gradient method [9]. 2.2. Transient transfection of P. falciparum All transfections used the plasmid pHC1-CAT [4], which was a gift from A. Cowman, and was prepared using the QiaFilter Maxiprep kit (Qiagen). Transient transfection was carried out by a modification of the method of Wu et al. [3]. Prior to transfection 50 g plasmid was diluted into 200 l incomplete cytomix (120 mM KCl, 5 mM MgCl2 , 0.15 mM CaCl2 , 2 mM EGTA, 25 mM HEPES in 10 mM potassium phosphate buffer, pH 7.6), which was then mixed gently with an equal volume of erythrocytes containing early ring stage parasites. Except where otherwise stated, the mixture was electroporated in a 0.2 cm cuvette at 300 V, 950 F using
a Bio-Rad Gene Pulser II and transferred immediately to 15 ml pre-warmed medium. All cultures were then gassed and incubated at 37 ◦ C. 2.3. Extracting cellular material for CAT assay Transfectants were cultured for 40 h, by which point the majority of parasites were segmenting schizonts, and then harvested as described by [3]. Parasitized erythrocytes were first washed with phosphate-buffered saline (PBS) by centrifugation, and then resuspended in two pellet volumes of PBS containing 0.15% (w/v) saponin, to disrupt the erythrocyte plasma membrane. After 10 min at 37 ◦ C the cells were spun down again and the haemoglobin-containing supernatant removed. Parasite pellets were stored at −80 ◦ C if necessary. After thawing the cells were resuspended in 1 ml of TEN buffer (40 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, pH 7.6), spun down, and resuspended again in 120 l 0.25 M Tris–HCl, pH 7.6. Next the samples were subjected to three cycles of fast freeze-thawing by transferring them between dry ice and a 37 ◦ C water bath. After a final incubation at 65 ◦ C for 10 min (to inactivate deacetylases) the extracts were centrifuged at top speed for 2 min in a microcentrifuge, and the supernatant containing the CAT enzyme removed. 2.4. Measuring CAT activity The basic assay was modified from the method described by [3]. To a sample of P. falciparum cell extract was added 5 l of a 10 mg ml−1 acetyl-CoA solution (final concentration 500 M), and 0.25 M Tris–HCl, pH 7.6 to a volume of 123 l. Reactions were started by adding 2 l (50 nCi) 14 C-labelled chloramphenicol (Amersham Biosciences No. CFA754), and incubated at 37 ◦ C for a specified time. Purified CAT was purchased from Promega. For analysis by TLC, CAT assay products were first extracted by phase separation, adding 400 l ethyl acetate, vortex mixing for 30 s, and then spinning in a microcentrifuge at top speed for 2 min. The organic (upper) phases were transferred into new tubes, and the ethyl acetate evaporated off in a Savant Speed Vac Concentrator (Model RH 40-11). Once dry, the products were redissolved in 10 l ethyl acetate and spotted onto silica coated aluminium TLC plates (Whatman No. 4420-221), allowing spots to dry between each application. The plates were then placed in a glass chromatography tank with a 19:1 (v/v) mixture of chloroform: methanol mobile phase. After 45 min to 1 h plates were removed, dried, and placed overnight in a phosphorimager cassette. The following day the cassette was scanned using a Molecular Dynamics Storm 860 phosphorimager, the data being analysed with ImageQuant 5.0 software. For LSC analysis, CAT assay products were extracted by phase separation, adding 300 l mixed xylenes, vortex mixing for 30 s, and spinning in a microcentrifuge at top speed for 3 min at 4 ◦ C. The organic (upper) phases were trans-
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ferred to fresh tubes and back-extracted by adding 100 l 0.25 M Tris–HCl, pH 7.6, vortex mixing and centrifugation as before. The back extraction was repeated and then the organic phases were transferred to scintillation vials with 2 ml Ready-Safe scintillant (Beckman). The activity of the samples was then measured on a Beckman LS6000IC scintillation counter, with a program set to count 14 C for 1 min per sample. 2.5. Estimation of total protein concentration Protein concentration was estimated using the Bio-Rad Protein Assay, using a 96-well plate, microassay format as recommended by the manufacturers. Bovine serum albumin was used as a standard protein and absorbance readings were measured using a Dynex MRX-TC 2 plate reader.
3. Results 3.1. Liquid scintillation counting is a convenient alternative to thin layer chromatography A LSC protocol was adapted for use with P. falciparum, and compared with the established TLC method. A set of parasites was transfected with pHC1-CAT under varying electroporation conditions, grown for 44 h, harvested as described and assayed for CAT activity. Half of the CAT reaction products were extracted with ethyl acetate and separated by TLC, and the other half by extraction with mixed xylenes. Products were measured by phosphorimager analysis and LSC respectively. Both sets of data were plotted as a percentage of the product from a positive control reaction containing 1 U commercially purified CAT (Fig. 1A). All the samples tested gave very similar results and variability for both methods, demonstrating that they are interchangeable. The LSC data were a consistently lower percentage of the control (average 5.8 ± 3.4%; P < 0.001 by paired t-test) for all samples; in most situations this difference would have no importance, but it suggests that at high CAT activities phosphorimager detection may saturate slightly sooner than LSC. To assess the efficiency of xylene extraction in separating products from substrate, controls of the CAT assay mix without enzyme were examined (Fig. 1B). In the initial xylene extraction less than 5% of the total radioactivity partitioned into the organic phase, and after two back extractions this was reduced to below 0.1%. At this level the radioactivity was indistinguishable from the background with scintillation cocktail alone, showing that essentially no substrate was extracted with the acetylated products. 3.2. Long incubations lead to assay saturation A curve was plotted of the amount of product using purified CAT in the previously reported assay protocol, to assess
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the method’s suitability for quantitative studies. Purified enzyme was diluted to 1 U l−1 in assay buffer, and two serial 100-fold dilutions were made from this stock. From these, assays were set up using 10 different quantities of enzyme covering four orders of magnitude: 2, 1, 0.4, 0.1, 0.04, 0.01, 0.004, 0.001, 0.0004 and 0.0001 U enzyme in a 125 l reaction. Each assay was set up in triplicate and incubated overnight at 37 ◦ C. The following day, the products from half of each reaction were measured by liquid scintillation counting. As shown in Fig. 2, the resulting curve was not linear. For enzyme activities below 0.001 U the radioactivity of the products was at background levels, while at higher enzyme activities saturation was observed in the assay. The only part of the curve that approximated to linearity was from 0.001 to 0.01 U (Fig. 2C), which is an extremely restricted linear range. The assay was saturating probably because one or both of the substrates was being used up. Each reaction contained about 100 000 counts per minute (cpm) (Fig. 1B) and with 2 U of CAT nearly 50% of the chloramphenicol had been acetylated by the end of the assay (Fig. 2, data for half the reaction). As noted in the introduction, chloramphenicol was not present in excess, so the substantial drop in substrate concentration at high CAT activities was bound to result in reduced product. 3.3. Effect of cell extract In preliminary experiments to improve the CAT assay, purified enzyme gave maximum acetylated product after 1–2 h incubation, and the amount of product dropped if incubated overnight. In contrast, the acetylated chloramphenicol produced when CAT-containing extracts from transfected parasites were used in CAT assays continued to increase when the extracts were incubated overnight (data not shown). This suggested that something in the P. falciparum extract stabilises the enzyme or its product. To test this hypothesis, P. falciparum cell extract was prepared from untransfected parasitized erythrocytes by the method used to obtain CAT, and verified to have no detectable CAT activity. Assays were then set up with 0.1 U purified CAT and 0, 50 or 100 l cell extract, the last sample imitating the conditions when CAT was extracted from P. falciparum. As shown in Fig. 3A, saturation occurred by 1 h, with no further increase in products by 2 h. However, after overnight incubation the amount of product dropped significantly, unless cell extract was present. A similar effect was observed in a 2-h time course with 0.01 U enzyme (Fig. 3B). In the presence of 100 l cell extract the amount of product increased linearly with time, whereas without cell extract the activity flattened out after about 1 h and then dropped on continued incubation. A reduced amount of cell extract (10 l per 125 l reaction) had an intermediate effect: the activity climbed throughout the 2-h time course, but the rate dropped with time.
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Fig. 1. (A) Ring stage parasites were transfected under a variety of conditions, harvested, and CAT assay reactions set up as described. After overnight incubation, the reaction mixtures were each split in half. The acetylated products from one half were separated and quantified using TLC and phosphorimaging (grey bars), and from the other half by xylene extraction and LSC (white bars). Results are expressed as a percentage of the control reactions, which contained 1 U purified CAT. Transfection conditions were: (a) 2000 V, 25 mF; (b) 280 V, 960 mF; (c) 250 V, 960 mF; (d) 300 V, 960 mF. Error bars show standard deviation of triplicate samples. (B) In all samples 50 nCi of d-threo-[dichloroacetyl-1-14C]chloramphenicol were diluted to a final volume of 125 l in 0.25 M Tris–HCl buffer, pH 7.6. Activity was measured after no extraction (Total counts), extraction with mixed xylenes (Extracted), one back extraction of the mixed xylenes using 100 l 0.25 M Tris–HCl buffer (Extr. + Back Extracted) or two such back extractions (Extr. + 2× Back Extracted). Error bars show standard deviation of duplicates.
The decrease in measured activity after extended incubation shows that neither the enzyme activity nor the products are entirely stable at 37 ◦ C. Product breakdown alone would produce an equilibrium when the rate of breakdown matched the rate of product formation, causing the measured activity to reach a plateau over time, but not to decrease until the substrate is exhausted. Under the conditions used in Fig. 3B, substrate was still abundant by the end of the experiment. Similarly, if the enzyme was deteriorating but the products were stable, the measured activity would reach a plateau but not decrease.
P. falciparum cell extract stabilised both enzyme activity and products. The amount required to give maximum CAT activity was measured by including different amounts of cell extract in assays of 0.01 U CAT incubated for 65 min. The effect of parasite cell extract reached a maximum at a concentration of 84 g of protein per ml, with a further 3-fold increase in concentration conferring no additional gain in activity (Fig. 4). Parallel assays were set up using the same concentration of bovine serum albumin (BSA). Including BSA in the assay also increased the amount of product in a dose-dependent fashion, but even at the highest
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Fig. 2. A wide range of enzyme activities were obtained by diluting commercially purified CAT and adding them to a standard CAT assay mixture. Reactions were incubated overnight at 37 ◦ C, and the acetylated chloramphenicol produced measured by xylene extraction and liquid scintillation counting. Error bars are standard deviation of triplicate samples: (A) entire data range; (B) expansion of shaded area of (A); (C) expansion of shaded area of (B). The points in the linear region of the curve are picked out with filled diamonds.
concentration tested the yield did not approach the maximum activity seen with cell extract. All subsequent reactions using purified CAT were supplemented with P. falciparum cell extract at a final concentration of 84 g of protein per ml. 3.4. Developing a quantitative protocol for CAT assay Long incubations may lead to product saturation as the chloramphenicol substrate is used up, and loss of product occurs—although this loss is ablated in the presence of cell extract. To solve these problems we measured the initial rate of the reaction, rather than the amount of product after a certain incubation period. This method is widely used in enzyme kinetics because it avoids the problems of enzyme instability and product saturation; in practice, the initial rate is often taken as the reaction rate before 10% of the substrate has been used up. A wide range of CAT concentrations was freshly made in triplicate in an assay mix supplemented with P. falciparum
cell extract. A time course at each concentration was carried out, with typical data shown in Fig. 5A. The initial rate of reaction for each curve was calculated by linear regression analysis of all the points in the linear section of the curve. These rates, in cpm min−1 , were plotted against CAT activity in U (Fig. 5B). Owing to the broad range of data points, the values were plotted on a logarithmic scale. The relationship between initial rate and enzyme concentration would be predicted to be a straight line of the form: Vi = M[E]T Where [E]T is the total enzyme concentration, Vi is the measured initial rate of reaction, and M is the gradient of the graph in Fig. 5B. M was calculated separately for the values at each data point and the mean of these values was 12 193 ± 2142 cpm min−1 U−1 . The line of best fit computed from this value is plotted on Fig. 5B, with the faint lines either side indicating the standard deviation.
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Fig. 3. CAT assays were set up using known amounts of purified CAT in the presence of varying amounts of P. falciparum cell extract as indicated. Total volume of all assay reactions was 125 l. (A) Reactions contained 0.1 U commercially purified CAT and incubated at 37 ◦ C for 1, 2 or 16 h before stopping the reaction by xylene extraction of the products. Error bars show the standard deviation of duplicates. (B) Time course of reaction for 0.01 U purified CAT. At each time point reactions were stopped by xylene extraction. Error bars show the standard deviation of duplicates. The line of best fit to the ‘100 l cell extract’ dataset was calculated by linear regression analysis and is plotted on the graph.
3.5. Comparison of old and new CAT assay protocols in the transient transfection assay The new, quantitative protocol was compared with the previously published method firstly by studying the effect of initial parasitaemia on transfection efficiency in a transient transfection assay. Starting with a P. falciparum culture containing early ring stages at about 12% parasitaemia, aliquots were mixed with unparasitized erythrocytes to give a range of parasitaemias, which were measured by counting thin blood smears. At each parasitaemia, four aliquots of cells were electroporated with 50 g pHC1-CAT, cultured for 40 h, harvested, and assayed for CAT activity. Two aliquots were then assayed using the TLC-based published method (Fig. 6A), and two using the new LSC-based protocol (Fig. 6B). To apply the new protocol, the cell extract from each transfected cell culture was used to prepare three 30 l aliquots for CAT assay. The total protein concentra-
tion of each extract was estimated using the BCA assay and each sample was supplemented with cell extract from untransfected parasites to the same total protein concentration, as required. The three assay mixtures from each extract were then incubated for three different times, stopping the reaction each time by xylene extraction of the products. The amount of product at the three time points was used to calculate the initial rate of reaction, and these rates were compared with the standard curve (Fig. 5B) to give a measure of the total CAT activity at each parasitaemia (Fig. 6B). The differences between the two datasets are immediately apparent. Using the published assay method, it appears that the relationship between parasitaemia and CAT activity is described by a saturation curve across the whole range. However, the quantitative protocol shows that activity actually increases linearly across the range until at least 12% parasitaemia; previous observations (data not shown) found that at higher densities the parasites become stressed and
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Fig. 4. P. falciparum cell extract obtained from 2 × 109 erythrocytes infected by schizonts at 10% parasitaemia was found to contain 350 mg ml−1 total protein. Bovine serum albumin (Sigma) was diluted to the same concentration in 0.25 M Tris–HCl, pH 7.6. 0.01 U commercially purified CAT enzyme was freshly diluted into reaction aliquots of 125 l, which included 0, 10, 30, or 90 l P. falciparum cell extract or BSA as indicated. All reactions were incubated for 65 min at 37 ◦ C, and then stopped by xylene extraction followed by liquid scintillation counting of the acetylated products. Error bars show the standard deviation of triplicate samples.
some die under the culture conditions used. The quantitative method also gives a measure of CAT activity, which the old protocol cannot. A similar, although less marked effect, was observed when the amount of plasmid used in the transfection was varied (Fig. 7). In this case, rings at 2% parasitaemia were used to minimise the saturation due to parasitaemia observed in Fig. 6A. Again the data obtained by the published method suggests that when more than 100 g plasmid is used, transfection efficiency rapidly saturates. However, the quantitative protocol suggests that while efficiency does begin to saturate at this high plasmid concentration, the effect is much less dramatic. Additionally, this experiment allows the lower detection limit of the two methods to be compared; neither method detected activity significantly above background when 2 g plasmid was used, but the quantitative protocol detected activity from 5 to 10 g plasmid where the published method did not.
4. Discussion The experiments reported here demonstrate that the CAT assay previously described for use with P. falciparum is qualitative, but not quantitative; that is, it can show that under the same conditions sample A contains more CAT than sample B, but not how much. Several refinements were made to the assay, which should be valuable for the quantitative analysis of gene expression and regulation in P. falciparum. At the final stage of the assay, separation and measurement of CAT reaction products was streamlined by using xylene extraction and LSC instead of TLC and phosphorimaging. This technique is at least as accurate, repro-
ducible and sensitive as the established method, and is less laborious. During the development of this assay it was observed that purified CAT enzyme is stabilised by the addition of P. falciparum cell extracts during prolonged incubation at 37 ◦ C. Similarly, Mandel [10] observed that purified CAT subjected to the 65 ◦ C preheating step to inactivate cell-derived deacetylases, lost at least 50% of its activity. Therefore if purified CAT is used as a standard for quantitating assay data it must be stabilised in some way. BSA is often added to enzyme reactions to increase stability, but in this study cell extract from untransfected P. falciparum was significantly more effective and it replicates more closely the conditions in the samples under test. The assay products are also stabilised by the presence of P. falciparum cell extract. It has previously been reported that cellular material from Saccharomyces cerevisiae increases activity in the CAT assay in a concentration dependent manner, both for endogenously produced and for purified bacterial enzyme [11]. This study also found that desalting by gel filtration greatly reduced the effect of the cell extract, implicating small molecules as being largely responsible. From assays using known amounts of CAT it was found that the established assay is linear only across a small range of enzyme concentrations. At high CAT activities the long incubation time leads to saturation of the assay by chloramphenicol depletion. Levels of CAT sufficient to cause this problem were generated in transient transfection experiments, as shown in Fig. 6A. Saturation of the assay makes it misleading; for example from Fig. 6A one might conclude that the difference in transfection efficiency at 5 or 10% parasitaemia is <20%. However, Fig. 6B shows that the difference is much greater than this. Similarly Fig. 7 shows
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Fig. 5. Initial rates of reaction were measured for a range of quantities of purified CAT enzyme and used to generate a standard curve. (A) Typical data showing time courses for 0.01, 0.003 and 0.001 U CAT as indicated. All reactions contained 30 l P. falciparum cell extract in a total volume of 125 l. Error bars show standard deviation of triplicate samples. Lines of best fit were calculated by linear regression analysis of all points lying within the initial linear phase, and plotted on the graph. (B) The gradients of the lines of best fit were taken as measures of the initial reaction rate and plotted against enzyme activity, with error bars showing the standard error of these values. At each point on this graph, y/x was calculated to obtain an estimate of the gradient of the standard curve. The mean of these estimates was found and used to plot a line of best fit to the data (solid line). One standard deviation of this line of best fit is plotted above and below the line itself (dashed lines).
that using plasmid in higher quantities than normally used in transient transfections (typically 50 or 100 g [3–5]) gives a greater increase in transfection efficiency than indicated by the published method. These differences could be important when, for example, trying to boost detection of activity from a weak promoter. In the latter example the new protocol also detected smaller amounts of CAT than the published method. A likely reason for this is that when a very small amount of CAT has been produced, an overnight incubation is long enough for it to degrade. We would expect the stabilising effect of P. falciparum cell extract to solve this problem, but in this instance using a lower parasitaemia (2% rather than 10%, to avoid the saturation seen for the old protocol in Fig 6A) and dividing the extract in two (half for each assay method) meant that the cell extract in the samples measured by the published method would have been one tenth of its normal
amount, which, as shown by the middle dataset in Fig. 3B, gives only partial stabilisation of the assay. It is expected that a higher parasitaemia would give improved detection of the lowest amounts of plasmid tested but greater saturation of the assay when using higher amounts. The results shown here contrast with those of Wu et al. [3], who detected CAT activity following transfection with as little as 1 g of plasmid. In that study the starting parasitaemia was 10%, not 2%, and CAT expression was driven by the hsp86 5 upstream region, which is believed to be stronger than the Calmodulin promoter used in pHC1-CAT. A fully quantitative assay protocol was developed for the reporter enzyme CAT, following transfection of the gene into P. falciparum. This assay will be of considerable value as a positive control for transfection, in comparing transfection efficiencies, and in characterising promoters. These authors have since used the improved assay to study the activities
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Fig. 6. Effect of parasitaemia on transfection efficiency measured by old and new protocols. (A) Cell extracts were prepared from duplicate cultures of transiently transfected erythrocytes as described in Section 2. One-hundred microliters of each extract were included in CAT assay reactions which were incubated overnight before xylene extraction and liquid scintillation counting. The chart shown includes data from two different experiments carried out on different days on different parasite cultures. Error bars show the standard deviation of between two and four samples. (B) Cell extracts from duplicate cultures were assayed for total protein concentration. CAT assay reactions were then set up containing 30 l extract from one of the transfected cultures, supplemented with untransfected P. falciparum cell extract to give a total protein concentration equal to that of the 12% parasitaemia cultures. For each culture three assays were set up which were incubated for 0, 1 or 2 h before extraction with mixed xylenes and liquid scintillation counting. Linear regression analysis was used to calculate the initial rate of reaction for each culture, which was compared with the standard curve in Fig. 5B to obtain a measure of the CAT activity present (plotted above). Error bars show the standard deviation of the values for initial rate estimated at each time point. A line of best fit was plotted by linear regression.
Fig. 7. Effect of amount of plasmid used on transfection efficiency measured by old and new protocols. A range of quantities of pHC1-CAT from 2 to 200 g were diluted into 200 l incomplete cytomix and transfected into 200 l aliquots of packed rbcs parasitized with 2% rings as described in the methods section. On extracting cellular material for CAT assay, each extract was divided in half, one half of the extract being assayed by the old protocol and one by the new protocol. Activities from the old protocol (䉫) were measured by liquid scintillation counting and are plotted against the left-hand axis. Samples from the new protocol were incubated for 0, 135 and 270 min before extraction and scintillation counting. Linear regression analysis was then used to estimate the initial rate of reaction and compare this with the standard curve in Fig. 5B to give values (䊏) that are plotted against the right-hand axis. Error bars show the standard deviation of duplicate or triplicate transfections.
of a range of promoters (manuscript in preparation). The new protocol is somewhat more labour intensive than the published one, as it requires at least three time points to be measured for each sample under test. However, this requirement is balanced by the shorter incubation time and simpler
separation and quantification of products, so that the entire assay can be completed in 1 day rather than 3 days. Furthermore, it gives an absolute measure of the CAT activity in a sample, rather than just a relative one; e.g. after transfection at 10% parasitaemia, 0.025 U CAT are generated from
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the calmodulin/PcDT promoter in pHC1-CAT. If this measure were combined with an accurate measure of transfection efficiency, the amount of CAT produced per transfected parasite could be calculated. Another reporter gene widely used in Plasmodium transfection studies is firefly luciferase, which can be quantitated by producing a standard curve in parallel with each experiment [12]. More recently a second luciferase, from Renilla regiformis, has also been applied to P. falciparum [13]. The luciferase assay is quicker than the CAT assay, although the simplification of the detection method described here reduces the difference. In some situations the CAT assay may be preferable as the luciferase gene product is relatively short-lived. For example, in a time course experiment luciferase produced from a promoter with a fairly small expression window might decay before the next measurement. To date the relative sensitivities of these two reporter genes have not been directly compared in P. falciparum. Whatever the relative advantages and disadvantages of each, having both of them available opens up a range of experiments that are not possible with one alone, such as dual labelling by fusing them to two different proteins, or using one to measure expression while using the other as an internal control. The observations made in this study illustrate the importance of thoroughly characterising an assay system before applying it to biological questions. Further improvements to this and other assays, as well as new alternatives such as commercially available fluorimetric and ELISA-based CAT assays, should be investigated, as they would be of significant benefit to the field.
Acknowledgements Thanks to M. Grainger for providing parasites and assistance with parasite culture, to A. Cowman for the gift of the pHC1-CAT plasmid, and to M. Hensmann for useful discussion. S.J.L. was in receipt of a MRC Studentship.
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