Cloning of Leishmania major carbonic anhydrase 2 into pET28a vector
Leishmaniasis is a vector-borne (sandfly) parasitic disease caused by obligatory intracellular parasite of genus Leishmania. It affects ~97 countries around the globe including India. When sandfly takes a blood meal Leishmania promastigotes came inside mammalian host and readily engulfed by macrophages. Within macrophages they reside and multiply within acidic phagolysosomal compartment. How Leishmania survives in such harsh acidic environment is yet to be solved. Carbonic anhydrases are group of ubiquitous metalloenzyme which catalyzes the conversion of carbon dioxide to bicarbonate and vice-versa. Literature review shows that L. major using their two carbonic anhydrases viz. LmCA1 (cytosolic) and LmCA2 (cell surface) maintain their neutral intracellular pH. Among these two CA, LmCA1 has already been purified and characterized however; LmCA2 is still to be purified and characterized. The broad aim is the purification using bacterial expression system followed by biochemical characterization of LmCA2. For the same, the first objective is cloning of LmCA2 (LmjF.28.0480) into pET28a vector in Escherichia coli DH5α cells and transformation of the clone into BL21 (DE3) E coli cells for protein expression.LmCA2 was amplified by using a pair of primer viz. forward primer and reverse primer with suitable restriction enzyme sites incorporated into primer, using Phusion polymerase, using L. major genomic DNAas template.LmCA2 and pET28a were digested by using two restriction enzymes viz. EcoRI-HF and NotI-HF. LmCA2 with pET28a were ligated by using T4 DNAligase. The ligated mixture is transformed into competent E. coli DH5 alpha cells by chemical transformation. The colonies were screened by colony PCR method and verified by restriction digestion using same enzyme used for cloning. The clone will be now transformed into E. coli BL21 (DE3) strain and will be used for protein expression. Successful cloning helps in the generation of desired protein i.e. LmCA2 via induction by using IPTG. Successful purification of the protein from system will help in generation of antibody required for different biochemical studies.
Keywords: Leishmania, LmjF.28.0480, Carbonic anhydrase, Bacterial protein expression.
Leishmaniasis is a vector borne protozoan parasitic disease caused by Leishmania spread by the bite of infected (with Leishmania) female phlebotomine sand flies of the genera Phlebotomus & Lutzomyia. There are about more than 90 species of vector responsible for transmitting Leishmania parasite. Leishmaniasis is classified as neglected tropical disease (NTD) and affects about 90 countries in the tropics, subtropics and southern Europe including India, South America, Southern Europe, North Africa etc. There are more than 20 species of Leishmania responsible for three different types of leishmaniasis viz. Cutaneous leishmaniasis, Visceral leishmaniasis and Mucocutaneous leishmaniasis. Post kala-azar dermal leishmaniasis is seen as a continuation of Visceral leishmaniasis which occurs 6 months to 1 or more years after cure of the disease.
|Leishmaniasis type||Leishmania species|
|Cutaneous leishmaniasis||L. major, L. tropica, L. mexicana|
|Visceral leishmaniasis||L. donovani, L. infantum, L. chagasi|
|Mucocutaneous leishmaniasis||L. braziliensis|
|Post kala-azar dermal leishmaniasis||L. infantum, L. donovani|
Leishmania is an obligatory, intracellular, digenetic protozoan parasite. It completes its lifecycle between two morphological forms viz. extracellular, motile, elongated, and flagellated promastigotes (in sand-fly-Phlebotomus) and intracellular, nonmotile, circular amastigote (in vertebrates). Promastigotes are found in the alimentary canal of sand fly whereas the amastigotes are found in the mononuclear phagocytes and circulatory system of vertebrates.
The life cycle and transmission of Leishmania spp. are much the same regardless of the species or clinical effects.When a sand fly takes a blood meal from a vertebrate, it releases promastigotes in the place of bite. Then the promastigotes are enter into the circulatory system and engulfed by macrophages and contained is so-called phagosome. The phagosome, in turn, fuses with a lysosome to form a phagolysosome. These organelles contain various enzymes, responsible for hydrolysis of different organic compounds. Inside the phagolysosome promastigotes aretransformed into the amastigotes, multiply within it and release infection after cell brust (Fig. 1).
Phagolysosome is a cytoplasmic organelle, function by maintaining its internal envirnoment to low pH condition which modulate the host defence mechanism by inhibiting the growth of pathogens and also enhance the activity of hydrolytic enzymes requird for breakdown of different organic molecules. How Leishmania survives such hosile low pH condition is yet to be solved. A putative mechanism have been provided by Pal-Abbasi et al., 2017, where they showed carbonic anydrases of Leishmania are indispensible for pH regulation
Carbonic anhydrases are a group of ubiquitous metalloenzyme which catalyzes the conversion of carbon dioxide to bicarbonate and vice-versa. Carbon dioxide, a poorly water soluble gas, which may damage cellular components (e.g. membranes) if generated in exceedingly high amounts in a cell/tissue, whereas its conversion to water soluble ions bicarbonate and protons), may interfere with the pH balance of the cell through the generation of an acid (H +) and a buffering base (HCO3−). Thus, catalysts responsible for the rapid interconversion between these chemical species have evolved at least 6 times independently, in a very interesting example of convergent or divergent evolution phenomenon, with six CA enzymatic families known to date, the α-, β-, γ -, δ-, ζ –and η-CA (Supuran, 2016).
|Alpha||Vertebrates, Protozoa, Algae, cytoplasm of green plants and in many gram-negative bacteria|
|Beta||Gram positive and gram-negative bacteria, algae, chloroplasts of mono- as well as di-cotyledons, and also in many fungi and in some Archaea.|
|Gamma||Archaea, cyanobacteria, and most type of bacteria|
In many organisms this enzyme plays very crucial role in physiological processes linked with pH and CO2 homeostasis. Many CAs from various organisms (protozoa, bacteria, nematodes, etc.) are drug targets, science interfering with their activity (by inhibition or activation) leads to a pharmacological response.
Structure of CAs
All known CAs from the animal kingdom are of the alpha-type. Carbonic anhydrases are metalloenzymes and the apoenzyme is catalytically inactive when unbound with a prosthetic metal ion. The active center normally contains M(II) ions in a tetrahedral geometry, with the 3 amino acid residues as ligands, in addition to a water molecule/hydroxide ion coordinating the metal (fig 2). In case of alpha carbonic anhydrases three His amino acid ligands coordinate with Zn (II) with water molecule/hydroxide ion, acting as nucleophile in the Catalytic cycle (Supuran, 2016).
There are 16 different isoforms of mammalian alpha CA, based on different catalytic activity, subcellular localization and tissue distribution. There are five cytosolic isoforms (CA I, CA II, CA III, CA VII AND CA XIII), Five membrane bound isozymes (CA IV, CA IX, CA XII, CA XIV and CA XV), two mitochondrial forms (CA VA AND CA VB), and a secreted CA isozyme (CA VI).
2. LITERATURE REVIEW
CAs are emerging as novel drug target in different pathogen. The indispensible role of Leishmania CAs in adaptation to harsh phagosomal condition has already been reported by Pal-Abbasi et al., 2017. They reported that Leishmania major possess two carbonic anhydrases viz. LmCA1(cytosolic) and LmCA2(cell surface), which protects it from intracellular acidosis. They also showed that the pharmacological inhibition of CA activity causes the intracellular acidoisis of Leishmania. It appears that Leishmania CAs can be targeted for development of new therapeutic. Pal ,2017 .
In 2013, Pan et al. reported the successful cloning and characterisation of alpha carbonic anhydrase from Trypanosoma cruzi which is a causative agent of American Trypanosomiasis or chagas disease Pan, et al ,2013.
In 2013, Syrjanen et al. have successfully cloned, purified and characterized a β carbonic anhydrase from Leishmania donovani chagasi responsible for Leishmania, Syrjänen, et al ,2013 . But the α-carbonic anhydrase of Leishmania is yet to be purified and characterized biochemically.
To clone Leishmania major carbonic anhydrase 2 (LmjF.28.0480) into pET28a vector.
Transformation of the clone into E coli protein expression strain (BL21 DE3) and check for induction of transformed protein by using IPTG.
MATERIALS AND METHODS
Media and Buffers
M199 Media (Leishmania major culture media)
The components were added are as follows:
|M199 Powder||13.2 gm/L|
|Adenine||2.4 mL/L (from 0.1M stock)|
|Folic Acid||.18 gm/L|
|Penicillin, Streptomycin (100 units)||6 ml/L|
|Gentamycin (40mg/ml)||750 µL/L|
Volume adjusted with distill water, the components were mixed, pH adjusted to 7.2 and filtered with 0.22 µm membrane filter.
Luria broth and agar (Bacterial media)
|Yeast extract||5 g/L|
The components are mixed, then autoclaved. To prepare agar plate 1.5% Agar-agar (15 g for 1 L) was added. Whenever required the media was supplemented with Kanamycin (concentration 50 µg/ml)
50X and 1X TAE buffer
|Glacial Acetic acid||57.04 mL/L|
Adjusted the pH to 8.
1 Liter 1X TAE was prepared from 50X stock, by adding 20ml 50X TAE and adjusting the volume to 1 liter by water.
1% agarose gel
Agarose - 1 gm
1X TAE - 100mL
EtBr - 5 µL
Components Resolving Gel (12%-10mL) Stacking Gel (5%-10mL)
MiliQ Water - 3.3mL 5.6mL
1M Tris HCl pH 8.8mL 2.5mL
0.5M tris HCl pH 6.8mL 2.5mL
30% Acrylamide with
0.8% Bisacrylamide 4 mL 1.7mL
10% SDS 100 µL 100 µL
10% APS 100 µL b 100 µL
TEMED 10 µL 10 µL
Deaerate after addition of APS and then TEMED was added.
Coomassie staining solution
Coomassie Brilliant Blue- 1g/L
Methanol - 500 mL/L
Elix Water - 400 mL/L
Glacial Acetic Acid - 100 mL/L
Coomassie destaining solution
Methanol - 400 mL/L
Elix Water - 500 mL/L
Glacial Acetic Acid - 100 mL/L
Lysis buffer (for 1l)
|NaH2PO4||50 mM||6 g|
|NaCl||300 mM||17.4 g|
|NaH2PO4||50 mM||6 g|
|NaCl||300 mM||17.4 g|
|NaH2PO4||50 mM||6 g|
|NaCl||300 mM||17.4 g|
|Oligo||Sequence (5'-3')||Restriction site|
Genomic DNA isolation from L. major by phenol chloroform method.
2×107 a cell of L. major promastigotes is centrifuged at 100g, washed with 1 mL of 1X PBS. To it added 200 µL of Lysis buffer (0.1M Tris HCl pH-8, 1% SDS, 0.1M HCl, 10mM EDTA) along with Protein kinase K and then incubated at 550C for 2 hours. To it added 1 mL of Phenol: Chloroform. Centrifuge at 13,000 rpm for 15 minutes. The upper aqueous phase was collected in a separate tube and added equal volume of chloroform to the remaining. Centrifuged at 13,000 g for 15 minutes. The upper aqueous phase was collected and added 1/10th volume of 3M Sodium Acetate and 2 volumes of absolute alcohol to the aqueous phase. Centrifuged at 13,000g for 10 minutes. The pellet was washed with 70% alcohol. Finally, the pelted was resuspended in 200 µL of nuclease free water.
Amplification of LmCA2 open reading frame (1806 bp)
|Nuclease free water||33.5µL|
|5X GC buffer||10 µL|
|10µM Forward primer||2 µL|
|10µM Reverse primer||2 µL|
|gDNA of L. major||1 µL|
|Phusion Polymerase (2units/ µL)||0.5 µL|
Cycle repeated for 30 timesPCR protocol
|Initial denaturation||980C||30 seconds|
|Extension||720C||1 min 25 seconds|
|Final extension||720C||10 minutes|
Gel extraction by using Qiagen kit
The excised gel was weighed and 3 volumes of QG buffer was added to it and incubated at 50˚c until the gel was dissolved. Added 1volume of isopropanol to it. Then transformed into a spin column. Then it was centrifuge at 13,000g for 1 minute. To it added 750 µL of PE buffer and incubated for 2 minutes. Centrifuged at 13,000g for 1.5 minutes. The column was transferred to a 1.5 ml centrifuged tube. To it added 30 µL of NFW and incubated at room temperature for 5 minutes. Centrifuged for 5 minutes at 13,000g. The DNA was stored in -20°C.
Isolation of plasmid pET28a from E. coli-DH5α
Plasmid were isolated using commercial kit using manufacture’s protocol. E. coliDH5α cells containing pET28a was grown in kanamycin (50 µg/mL) containing LB Medium and incubated it at 37°C for16 hours. The bacterial culture was taken in a tube and centrifuged at 3000g for 5 minutes and the pellet was resuspended with 250 µL resuspension buffer (R3 containing RNase). Added 250 µL lysis buffer (L7) to it and incubated at room temperature for 5 minutes. To it added 350 µL of precipitation buffer and mixed gently and centrifuged at 13,000 g for 10 minutes. The supernatant was transferred to a spin column and centrifuged at 13,000 g for 1 minute. Added 750 µL of wash buffer (with ethanol) to the column and centrifuged at 12,000 g for 1 minute. The spin column was transferred to a1.5 ml tube and 30 µL of NFW was added to it. Incubated for 5 minutes and centrifuged at 12,000g for 5 minutes. Finally, eluent contains the plasmid pET28a.
Restriction digestion of plasmid pET28a and LmCA2 insert
|Component||Plasmid (pET28a)||Insert (LmCA2)|
|NFW||32 µL||13 µL|
|DNA||11 µL||30 µL|
|10x Cutsmart buffer||5 µL||5 µL|
|EcoRI-HF (10units/µL)||1 µL||1 µL|
|NotI–HF (10units/µL)||1 µL||1 µL|
|50 µL||50 µL|
All the components ware mixed and incubated at 37˚C for 1 hour.
|NFW||1 µL||7 µL|
|T4 ligase||2 µL||2 µL|
|Plasmid (pET28a)||10 µL||10 µL|
|T4 ligase||1 µL||1 µL|
|20 µL||20 µL|
The ligation mixture was incubated at 16°C for 16 hours.
10 µL of ligation mixture was added to 100 µL of chemically competent cells (E. coli DH5α) (provided by the laboratory) and incubated in ice for 40 minutes. Heat shocked at 42˚C for 45 seconds followed by cold shock for 5 minutes. To it added 500 µL of LB medium without antibiotic and incubated at 37˚C for 1 hour. The cells are centrifuged at 3000g for 5 minutes. 500 µl of the supernatant was discarded and the pellet was resuspended in 100 µL of media and plated on Kanamycin containing agar plate. Incubated at 37˚C for 16 hours.
Colony PCR to screen for clone
4 random colonies were selected from pET28CA2DH5α agar plate for colony PCR and master plate. Colony PCR was done by using same forward and reverse primer using DreamTaq reaction protocol mentioned above. The colonies were directly mixed in reaction mixture. The amplified products were run in 60 ml 1% agarose gel. From this we conform that how many of the colonies were positive and then choose these colonies from master plate for restriction digestion.
Isolation of plasmid from putative positive colonies
pET28ACA2plasmid was isolated from positive colonies by using Invitrogen kit using the same protocol mentioned above.
PCR confirmation of pET28aCA2 clone
It was carried out using the same protocol as above.
Restriction digestion confirmation of pET28aCA2 clone
Restriction digestion were performed using the following protocol:
|NFW||33 µL||33 µL|
|DNA (pET28Aca2)||10 µL||10 µL|
|Cutsmart buffer||5 µL||5 µL|
|EcoRI-HF (10units/µL)||1 µL||1 µL|
|NotI-HF (10units/µL)||1 µL||1 µL|
|50 µL||50 µL|
All the components ware mixed and incubated at 37˚C for 1 hour.
Transformation OF pET28aCA2 construct into E. coli BL21DE3 cells
1 ul of pET28aCA2 plasmid was added in 100 µL of chemically competent cells (E. coli BL21(DE3)) (provided by the laboratory) and incubated in ice for 40 minutes. Heat shocked at 42˚C for 45 seconds followed by cold shock for 5 minutes. To it added 500 µL of LB medium without antibiotic and incubated at 37˚C for 1 hour. The cells are centrifuged at 3000g for 5 minutes. 500 µl of the supernatant was discarded and the pellet was resuspended in 100 µL of media and plated on Kanamycin containing agar plate. Incubated at 37˚C for 16 hours.
Checking for induction of protein CA2 by using IPTG
5mL primary culture of LmCA2pET28a BL21 (DE3) was prepared and50 µL (1%) of it was added to 5mL LB Medium (with kanamycin). Incubated at 370C up to OD 0.5. To it 5 µL of IPTG was added and incubated at 370C.1mL sample was collected after 3h, 4h, 5h, 6h, and 7h and centrifuged at 3000g for 5 minutes. Pellets were resuspended with 100 µL of 1X solubilizer and heated at 1000C for 5 minutes.
Solubility check for LmCA2
5mL primary culture of LmCA2pET28a BL21 (DE3) was prepared and 50 µL (1%) of it was added to 5mL LB Medium (with kanamycin). Incubated at 37°C up to OD 0.5. To it 5 µL of IPTG (also 5 µL of 0.5M ZnSO 4 added at the mean time if required) was added and incubated at 37°C for 7 hours and 20°C for 12 hours. After respective time periods the samples were collected and centrifuged at 3000 g for 5 minutes. Pellets were resuspended with 500 µL Lysis buffer and 0.5 mg of Lysozyme was added to it. Incubated in ice for 30 minutes and sonication (Pulse 15 second, Rest 10 second, Amplitude 100%, 3 cycles) of bacterial cells was carried out by using so nicator (20 kHz). 50 µL of Whole cell lysates (WCL) were collected and rest of the samples were centrifuged at 15000g for 30 minutes at 4°C. 50 µL of supernatant were collected and the pellets ware resuspended with 500 µL of lysis buffer. Then 50 µL of insoluble fraction were collected and to rest of it 0.24 gm of urea was added. Centrifuged at 15000g for 30 minutes at 4°C. Supernatant was collected and 50 µL of it was taken out as soluble fraction urea for running in the gel. Pellets are resuspended with equal volume of lysis buffer with 8 m urea as supernatant and 50 µL of it was kept as insoluble fraction urea. 2Xsolubiliser were added to all the samples like whole cell lysate, soluble fraction, insoluble fraction, soluble urea, and insoluble urea and heated at 100°C for 5 minutes.
RESULT AND DISCUSSION
Genomic DNA isolation from Leishmania major and PCR amplification of LmCA2
Since Leishmania genome are intron-less genomes, we planned to use genomic DNA as template for amplification of any gene sequence for protein expression. We successfully isolated gDNA for the same. Leishmania major carbonic anhydrase 2 is a membrane surface protein, it possesses a signal sequence towards N-terminal end for ER targeting, which is cleaved in secretory pathway of Eukaryotes. Bacteria are devoid of such system, so ignored the signal peptide region, and plan to clone the gene after signal peptide, instead of full length. So, amplification was done, from region 82bp to 1887bp (1805bp). Plasmid pET28a (with N-terminal 6X histidine tag) was used so that, the recombinant protein is made with N-terminal 6X his tag, which is planned to use for purification with Ni-NTA affinity chromatography.
After PCR amplification with Phusion polymerase of LmCA2 by using above mentioned oligoes the PCR product was run in 1% agarose gel and visible 1.8 kb band (fig 3) indicates correct amplification of desired LmCA2 gene sequence from Leishmania genome. The band was gel purified and used for further downstream processes.
Isolation of pET28a
Plasmid pET28a was isolated by using Invitrogen kit and then run in 1% agarose gel. Three bands in the agarose gel (fig 4) represents the purified isolation of pET28a. The concentration of plasmid was determined by using nanodrop and found to be 97.8 ng/µL of DNA.
Restriction digestion of plasmid and insert, followed by gel extraction and ligation
Restriction digestion pET28a and LmCA2 were done by using Eco RI-HF and Not I-HF restriction enzymes. Digestion products were run in 1 % agarose gel. Suitable bands i.e. one at 5.3 kb and another at 1.8kb, for pET28a and LmCA2 respectively in gel shows that proper digestion of LmCA2 and pET28a. Single 5.3 kb band indicates, good digestion of plasmid, indicating proper digestion. The digested product was extracted, and then set for ligation.
Transformation and screening of positive colonies by colony PCR
After transformation LmCA2pET28a of into DH5α competent cells. 25 colonies were observed in test plate, 5 colonies were observed in control plate. For colony PCR of 4 random colonies from LmCA2pET28a DH5α plate were performed by using same oligos. 1.8kb band for colony 1, 2 and 4 in 1% agarose gel shows that colony 1, colony 2 and colony 4 were appears positive colonies (Fig 5).
Confirmation by restriction digestion
Plasmid pET28ACA2 were isolated from colony 1 and 2 by using Invitrogen kit. Restriction digestions of recombinant plasmids were performed by using EcoRI-HF and NotI-HF restriction enzymes. Digestion products were run in 1 % agarose gel. Suitable bands i.e. one at 5.3 kb and another at 1.8kb, for pET28a and LmCA2 respectively in gel shows presence of insert of LmCA2 to pET28a (Fig 6).
PCR confirmation of CA2from plasmid pET28ACA2of colony 1 and 2
For further more confirmation, PCR amplification of plasmid pET28ACA2 of colony 1 and 2 were performed by using same oligos. The 1.8kb band was visible for of both the colonies in 1% agarose gel confirms the presence of insert inside the plasmid (Fig 7).
Induction of protein CA2 by using IPTG
The induced and uninduced samples were run in a 12% SDS_PAGE. Since the expression of LmCA2 in pET28a was under the control of lac operon system, the induction band was visible in case of induced culture in comparison to un induced one. In fig 8:- the 71kd for each sample of +I in comparison to –I, shows that the sucessful induction of LmCA2. Also there is saturation post 3 hours of induction.
Solubility check at different conditions
71 kd band is present in +I, not present in -I, indicating succesful induction. Lysing the cell, the whole cell lysate is separated into soluble (SF) and insoluble fraction (IF). Most of the induced protein is present in IF. IF was further attempted to be solublised in pressence of 8M urea.
The mixture again separated into Insoluble fraction with urea (IFu) and Soluble fraction with urea (SFu). The protein is still present in IFu, indicating it is not properly solubilized (Fig 9).
Further, we tried induce the protein in 20°C. The logic behind that, sometimes, protein aggregates are formed, because protein misfolding occurs, due to lesser time available for folding. So, lowering the temperature, actually slows down the metabolism, help in better folding. But again it is clear that, majority of the protein is still present in insoluble form (Fig 10).
Finally, solubilization was tried by inducing protein in presence of Zinc. Since zinc is required in active site, insufficient amount of zinc can lead to improper folding and aggregation. Zinc was provided in form of Zinc sulfate. But still, protein in present in insoluble form (Fig 11).
The cloning pf LmCA2 into pET28a is successful, as shown by PCR and restriction digestion data ofLmCA2pET28a. The construct has been successfully transformed into Bl21 (DE3). Induction band at 71 kd conclude that the sucessful induction also. The solubilisation and purification work is still under progress. Once purified, the protein will be used for biochemical characterisation and antibody generation.
I am extremely thankful to Dr Rupak Datta (Associate professor DBS, IISER, Kolkata) for his continuous inspiration, supportive guidance and constructuctive suggestions.
I would like to express a special thanks to IASc-INSA-NASI for giving me a scientific opportunity to work at a specialised laboratory.
I would like to extend a special word of thanks to Mr Arunava Seth and all my well-wishers for giving me a helping hand when ever needed and for making this project a grand success.
School of life sciences
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