Abstract

Abstract:
Dihydrofolate reductase (DHFR) is a ubiquitous enzyme found in all prokaryotic and eukaryotic cells. It is found to play a major role in synthesis of thymidine. With the help of NADPH as cofactor, it catalyses the reduction of DHF to THF. This reaction is important in the biosynthesis of nucleotidic bases of DNA. As blockage of the DHFR enzyme causes cell death because of DNA synthesis inhibition it is considered an excellent target for antitumor drugs. The catalytic reduction of DHF to THF in the presence of NADPH is also seen in malarial parasites. This reduction leads to synthesis of pyrimidine in plasmodium which makes DHFR an important target for therapeutic agents. Keeping these properties of DHFR in mind, by developing a recombinant DHFR system and studying its activity will open wide arena in the development of therapeutic drugs.
In this study, E. coli was induced with IPTG to produce recombinant GST-DHFR-His protein. The GST-DHFR-His protein was purified by affinity chromatography and size exclusion chromatography. The purified recombinant protein was visualised by SDS-PAGE and the enzyme activity was calculated by adding substrate (DHF) to the purified protein and reading the absorbance at 340 nm. It was found from our study that purified recombinant GST-DHFR-His protein ran at approximately 48 kD. The change in concentration of NADPH over the reaction course time was calculated to be 3.858 x 10-6 mol/min.

Introduction:
Dihydrofolate reductase or DHFR is an enzyme that reduces dihydrofolate, a folic acid derivative, into tetrahydrofolate (THF) by using NADPH as electron donor. DHFR enzyme is encoded by the DHFR gene in humans and is found on chromosome 5. DHFR is ubiquitous in prokaryotic and eukaryotic cells1. THF is a methyl group shuttle required for the synthesis of purines, thymidylic acid, amino acids and all essential for nucleic acids2. Deficiency in DHFR has been linked to megaloblastic anaemia, an anaemic disorder with larger-than-normal red blood cells, as well as cerebral folate metabolism disorders which can be treated with folic acid or Vitamin B12 depending on the severity of the symptoms3.
DHFR is responsible for the levels of tetrahydrofolate in a cell, and by inhibiting DHFR, the growth and proliferation of cells that are characteristic of cancer can be limited. Methotrexate is one such anticancer drug that inhibits DHFR4. Further studies in different inhibitors of DHFR can open a new arena to treat cancer more effectively.
Sometimes after repeated treatments with methotrexate, a cancer cells develop methotrexate resistance and will stop responding to the drug. When examined, some of these resistant cells show that the resistance was due to an increased copy number of DHFR genes. This gene amplification leads to increased levels of DHFR protein in the cell and therefore the ability to catalyze its reaction increases, thus producing nucleotides, even in the presence of methotrexate5.
DHFR also catalyses NADPH dependent reduction of dihydrofolate to tetrahydrofolate in malarial parasites. The synthesis of pyrimidine in plasmodium makes the enzyme an important target for therapeutic agents. Lately many drug-resistant strains have become common that are resistant to the once effective DHFR inhibitors pyrimethamine, sulphadoxine and methotrexate. The search for more effective drugs to stop the spread of malaria and cure the people effected by malaria is still under process by scientists6.
The main role of DHFR in the synthesis of nucleic acid precursors, along with its significant reactivity with THF analogs has made this enzyme a target of wide use in cancer therapy7 and treatment of malaria8. By developing a recombinant DHFR system and using it to study the enzymatic activity, it would help in developing anticancer and antimalarial drugs.
In this study, we induced the E. coli with IPTG to produce recombinant GST-DHFR-His protein. The GST-DHFR-His protein was purified by first using Nickel IMAC resin column and then was desalted using a size exclusion column to remove imidazole. The purified recombinant protein was visualised by SDS-PAGE and the enzyme activity was calculated by adding substrate (DHF) to the purified protein and reading the absorbance at 340 nm.
Materials and Methods:
GST-DHFR-His expression and purification:
The LB/amp plate was streaked with BL21-pDHFR bacterial strain and was incubated overnight at 37?. One colony from the plate was picked and cultured in LB/amp/20% glucose broth with shaking at 37? for 12-18 hours. The spectrophotometer was blanked using 1 ml LB/amp broth and the absorbance of the overnight culture (1:10 dilution) was read at 600 nm. A subculture was prepared with a starting OD600 of 0.3. A 1:3 dilution of the subculture was prepared and OD600 was measured.
100 mM of IPTG (an inducible promoter) was added to the subculture and was incubated overnight at 37? to let the E. coli produce the recombinant GST-DHFR-His protein. After 24 hours, the cells were pelleted by centrifugation. The cell lysis was performed by three freeze-thaw cycles in a lysis buffer containing lysozyme. The lysed cells were centrifuged at 16,000 x g for 20 minutes. The supernatant was transferred in a clean microcentrifuge tube and Lysis Buffer was added to the pellet and was sheared by pulling it through a gauge syringe.
GST-DHFR-His was purified using a Nickel IMAC resin. The Micro Bio-Spin column was centrifuged to remove the packing. The buffer collected was discarded and the column was washed with distilled water and equilibrated by adding equilibration buffer (20 mM sodium phosphate, 300 mM NaCl and 5 mM imidazole). The yellow tip closure was attached to the bottom of the column and soluble fraction of the lysate was added along with the clear top cap and the column was rotated at room temperature for 20 minutes on a rotator. The “Flowthrough”, “Wash fraction” and “Eluate” were saved after adding the wash buffer and elution buffer (250 mM imidazole) respectively and centrifuging the column.
The desalting size exclusion column (Bio-Gel P-6) was centrifuged to remove any excess packing buffer. The “Desalted Eluate” was saved after adding approximately 150 uL of eluate fraction into the column and centrifuging the column.
All SDS-PAGE samples were prepared by centrifuging the samples at 16,000 x g for 2 minutes. The pellets obtained after centrifugation were transferred to a labelled microcentrifuge tube, suspended in LSB and heated at 95? for 5 minutes. The sample was stored at -20? until ready to analyse via SDS-PAGE analysis.
All the centrifugation steps were done at 1000 x g for 2 minutes unless otherwise indicated.
DHFR Enzymatic Activity Assay:
DHFR enzymatic activity was measured indirectly by measuring the absorbance at 340nm. For the no substrate control reaction, 10 mM NADPH and 15 uL purified desalted GST-DHFR-His were added to the UV compatible cuvette containing 1X PBS. The absorbance was measured every 15 seconds for 150 seconds. A time vs absorbance graph was plotted, and the slope was calculated.
For the enzymatic reaction, to the UV compatible cuvette containing 1X PBS, NADPH and purified desalted GST-DHFR-His, 10mM DHF was added. The absorbance was measured every 15 seconds for 150 seconds. A time vs absorbance graph was plotted, and the slope was calculated.
The slopes obtained from No Substrate Control Reaction and Enzyme Reaction graphs were multiplied with 60 to get the change in absorbance at 340 nm/minute which is referred to as ?OD, control and ?OD, reaction respectively. The ?OD was calculated by subtracting ?OD, control from ?OD, reaction.
The enzyme activity of purified GST-DHFR-His was calculated using Beer’s Law. The change in concentration of NADPH over the reaction course time ?C (mol/liter/min) was calculated by dividing ?OD by ? (extinction coefficient for NADPH = 6220 M-1cm-1) and length of the cuvette (1 cm).
Results:
As it is seen that DHFR reduces DHF into THF with the help of NADPH1 and because of its significant reactivity with THF analogs, DHFR can be considered as a target in cancer therapy4 and treatment of malaria5. In this study, we purified and visualised the GST-DHFR-His protein and calculated the enzyme activity of purified GST-DHFR-His.
E. coli was used to produce the recombinant GST-DHFR-His protein by inducing with IPTG. The recombinant protein produced was lysed using lysozyme and was purified using a Nickle-IMAC resin column and a desalting size exclusion gel column to remove imidazole.
SDS-PAGE was run to visualize the purified recombinant protein in the gel using the Coomassie stain. The purified recombinant GST-DHFR-His protein was successfully visualised (Figure 1). It was found that purified recombinant GST-DHFR-His ran at approximately 48 kD. With each step of the lysis and purification process, the 48 kD become more prominent.
From the graph for No Substrate Control Reaction (Figure 2A), the slope was calculated to be 0.0003 and the change in absorbance at 340 nm/minute (?OD, control) was calculated to be 0.018. From the graph for Enzyme Reaction (Figure 2B), the slope was calculated to be -0.001 and the change in absorbance at 340 nm/minute (?OD, reaction) was calculated to be 0.006. The ?OD was calculated to be -0.024. Hence, the change in concentration of NADPH over the reaction course time (?C) was calculated to be 3.858 x 10-6 mol/min.
Discussion:
DHFR is a crucial enzyme in the mechanism of folate, which is linked to the production of thymidine9. It is found that DHFR catalyses DHF to THF in the presence of NADPH1. DHFR has found to be associated with cancer and malaria. By inhibiting the activity of DHFR using inhibitors like methotrexate, anti-cancer and anti-malarial drugs can be produced4,6. In this study, we successfully purified and visualised the GST-DHFR-His protein and calculated the enzyme activity of purified GST-DHFR-His.
E. coli (BL21 strain) was selected as recombinant protein host system to produce the recombinant GST-DHFR-His protein by inducing with IPTG. E. coli is used as the recombinant protein host system as it has a quick doubling time of 20 minutes. The bacteria are also inexpensive and easy to culture, induce and lyse the cell contents that makes it a perfect selection for this study. IPTG induces the expression of T7 ENA polymerase. In the absence of the inducer, there is no expression of the gene. A GST tag was added to the N-terminus of DHFR to increase its solubility and histidine sequence was added to the C-terminus of DHFR to allow easy purification by metal affinity chromatography. To determine if the cells have reached their mid-log phase, the absorbance of the culture is read at 600 nm. An OD600 of 0.6-1.0 is typically considered as the typical target for induction of IPTG.
After culturing the cells for 24 hours of induction with IPTG, the recombinant protein produced was lysed using lysozyme (an enzyme which digests the cell wall) along with freeze-thaw cycles. The lysed protein was purified using a Nickle-IMAC resin column. The histidine groups of the polyhistidine tag bind to the Ni++ groups on the resin. The recombinant protein is eluted out by the addition of imidazole which competes with the nickel binding sites. To remove excess salt and imidazole from the recombinant protein, a size exclusion gel column was used. The fractionation range of the gel used in these columns is 1,000 to 6,000 Da which means that the gel pores are large enough to allow molecules in the 1-6 kD range, like salts (imidazole, NaCl and sodium phosphate) and small proteins, to enter but larger molecules like GST-DHFR-His, with a predicted molecular weight of 52 kD, will be eluted out of the column.
To determine the purity of the purified recombinant protein, SDS-PAGE analysis is done using the Coomassie stain. The purified recombinant GST-DHFR-His protein was successfully visualised (Figure 1). It was found that purified recombinant GST-DHFR-His ran at approximately 48 kD. With each step of the lysis and purification process, the 48 kD become more prominent. According to various experiments conducted, it is found that purified GST-DHFR-His runs at approximately 43 kD which is quite close to the band (48 kD) that we observed. This could be due to some discrepancy in the purification step. There could be some inconsistency while desalting the recombinant protein. The yield of the recombinant protein can be increased by incubating the E. coli in the LB/amp/20% glucose broth for some extra time and to induce it with IPTG when the bacteria is in the active mid-log phase. By doing so, more amount of recombinant protein will be created and hence the yield will also increase.
By analysing the enzyme activity, it was seen that for No Substrate Control Reaction (Figure 2A), the change in absorbance at 340 nm/minute (?OD, control) was calculated to be 0.018 and for Enzyme Reaction (Figure 2B), the change in absorbance at 340 nm/minute (?OD, reaction) was calculated to be 0.006. The ?OD was calculated to be -0.024. Hence, the change in concentration of NADPH over the reaction course time (?C) was calculated to be 3.858 x 10-6 mol/min. It can be concluded that a good amount of enzyme activity was seen, and the recombinant protein can be used as a potential target for inhibitors to produce therapeutic drugs. By developing a recombinant DHFR system and using it to study the enzymatic activity, it would open new arenas to research and development of anticancer and antimalarial drugs.