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Summer Research Fellowship Programme of India's Science Academies

Development of strategies for purification of native and His-tagged mature form of Organophosphate Hydrolase

Harshita Kasera

Central University of Rajasthan, University Rd, Bandar Sindri, Rajasthan 305816

Guided by:

Prof. Siddavattam Dayananda

University of Hyderabad, CUC, Prof C. R. Rao Road, P O Central University, Gachibowli, Hyderabad, Telangana 500046

Abstract

The main aim of the project is to determine the molecular mass of the mature form of OPH (mOPH) in its native form. In order to determine the the true mass of mOPH the protein is needed in the purified form. The purification of OPH is performed by various chromatographic techniques, native PAGEand MALDI-TOF mass spectroscopy. This determines the actual size of the protein sample. The purification process takes longer than expected. To reduce the time needed to purify the mOPH or to directly purify by a one-step process His-tag coding sequence has to be cloned in frame with the opd gene at its 3’ end. We developed and optimized the strategies for the purification of the native and His-tagged mOPH.

Keywords: organophosphate hydrolases, pUC19+opd, pPHNS, purification

Abbreviations

OPH Organophosphate hydrolase

opd OPH degrading gene

OP organophosphate

TAT twin arginine transport

AchE Acetylcholine esterase

NaCl Sodium chloride

NaOH Sodium hydroxide

KCl Potassium chloride

CoCl2 Cobalt chloride

K2HPO4 Potassium phosphate dibasic

KH2PO4 Potassium phosphate monobasic

HCl Hydrochloric acid

MPT Methyl parathion

APS Ammonium persulfate

TEMED N,N’-tetramethyl ethylenediamine

SDS Sodium dodecyl sulphate

PAGE Polyacrylamide Gel Electrophoresis

TAE Tris Acetate EDTA

EDTA Ethylene diamine tetra acetate

FPLC Fast Protein Liquid Chromatography

CBB Coomassie Brilliant Blue

Wt wild type

Mut Mutant

RE restriction enzyme

PCR polymerase chain reaction

kb kilo base pair

bp base pair

Da Dalton

kD kilo Dalton

Introduction

 Organophosphate hydrolase

Organophosphates are esters of phosphoric acid with the general chemical structure being O=P(OR)3. They are widely used as a major component of pesticides as they inhibit the acetylcholinesterase activity leading to death of the insect. Acetylcholinesterase (AchE) is present in various species, thus the after effects of inhibiting this enzyme comes up as a major danger to all because it alteris the neural signal transmission pathway.

1.png
    Acetylcholinesterase inhibition by organophosphate

    The commercially used OP pesticides and insecticides includes parathion, malathion, methyl parathion, phosmet, fenithrothion, terbufos, diazinon and others. Prolonged exposure of OP can cause various health problems such as cardiovascular and respiratory diseases and even cancer. These agents of chemical warfare against pests increases the risk of diseases in humans. Its degradation rate is slow and its residues are observed in various crops and in the water running from the fields. The United States environmental protection agency has listed parathion as a potential carcinogen and the international agency for research on cancer has also listed tetrachlorviphos and parathion as a carcinogen and malathion and diazinon as probably carcinogenic to humans.

    Besides this, it is widely used in agriculture and thus the need to detoxify these carcinogenic chemicals is a major issue.

    Many bacterial species are being discovered as our friends in the process of degrading OP. They hydrolyse the ester bonds of the phosphotriesters. Species such as Sphingobium fuliginis ATCC27551 and Sphingopysix wildii are found to have the OP degradation activity (​Pandeeti et al 2012​) . Plasmids harboured by these species have the organophosphate degrading (opd) gene. S. wildii have pCMS1 (66kb) and S. fuliginis have ppPDL2 (40kb) plasmids that encode the 1098bp opd gene. The opd is also found to be a complex transposon and is a highly conserved gene in nature. It is a part of the Integrative Mobilizable Element (IME) (​Siddavattam et al 2003​).

    The opd codes for a phosphotriesterases, known as organophosphate hydrolases (OPH), a 365 amino acid long protein and is a dimeric metalloenzyme having zinc at its catalytic center. It is a membrane associated protein having a 29 amino acid long signal sequence peptide that directs it towards the inner membrane in a pre-folded conformation by the twin arginine transport (TAT) pathway. It has a TIM barrel fold in the protein quaternary structure. As observed in various studies, OPH is a 70kDa dimeric protein having 35kDa monomers (Gorla et al 2009)​.

    The OPH is a lipoprotein. Its signal peptide has an invariant cysteine residue at the signal peptidase cleavage site and also has a conserved lipobox motif. When the OPH producing cells were treated with the globomycin, a signal peptidase II inhibitor, it lead to the accumulation of a precursor of OPH (preOH) whole of the protein in the cytoplasm. When the cysteine residue is substituted by the serine residue, the OPH was found in the periplasm, confirming that OPH is a lipoprotein (Parthasarathy et al 2016)

    It is anchored to the membrane by using Diacyl glycerol that is further linked to myristic and oleic groups in the phospholipid bilayer (Parthasarathy et al 2017​)

    Some of the OPH encoding bacteria grow using the OP as the source of phosphate ions. The outer membrane has efflux proteins TolC and PstS that are the periplasmic components of the ABC transporter complex (PstABC) that is further involved in the phosphate ion transport system in these bacterial species. The OPH being bound to the inner membrane is shown to interact with this transporter complex and facilitate the phosphate ion transport, generated from OP compounds. It was observed that the opd null mutants of S. wildii were unable to grow when provided with methyl parathion as the sole source of phosphate.

    Thus, Organophosphates and the study of Organophosphate hydrolase is a major topic of research.

     Cloning of His-tag in opd

    pUC19 plasmid cloned with the wild type opd gene and the mutant opd gene were present in the laboratory. A mutation was caused at 82 amino acid residue by site directed mutagenesis and it has been changed from lysine to alanine to demonstrate that there is no effect of the change of the amino acid residue in the OPH protein. But the purification of the mutant OPH was a cumbersome and time taking process. So purification was not possible with this technique due to the change in the pI of the protein. Thus, it was planned to clone the His-tag to both the wild type and the mutant opd gene in order to fasten the purification process and to produce a similar affect on both of their structures.

    pUC19.png
      Vector map of pUC18/19
      pET23.png
        Vector map of pET23b

        Cloning of opd gene coding mOPH has been cloned

        His-tag cloned along with the wild type opd gene in the pET23 plasmid (Inc, 1998) as at the Nde1 and Xho1 fragment. Restriction sites were already present in the form of pPHNS without the signal sequence in the gene. The 3’ region of opd gene cloned in pET23b was taken as SalI-BglIE.coRI fragment. Thus, His-tag was digested from the pPHNS and was cloned into pUC19-OPDopd digested with similar enzymes SalI and BamHI. This strategy eliminates 3’ region of opd from pUC19 opd plasmid and inserts 3’ opd having His-tag coding sequence.

        Materials and methods

        Bacterial strains:

        Bacterial strains used

        Name

        Plasmid

        Gene

        Escherichia coli DH5α

        pUC19

        oOpd

        Escherichia coli BL21

        pET23b

        Opd

        Antibiotics and chemicals:

        Antibiotics and chemicals used

        Name

        Stock concentration

        Working concentration

        Ampicillin

        100mg/ml

        100µg/ml

        Lysozyme

        10mg/ml

        50µg/ml

        Chloramphenicol

        30mg/ml

        30µg/ml

        Lysozyme

        10mg/ml

        50µg/ml

         Growth media:

        The media were sterilized by autoclaving for 20min at 15lb/sq inch. When needed antibiotics were added to the media after cooling it to 40-50°C.

        Growth media used

        Name

        Composition

        Luria bertani (LB) broth

        Peptone 10g/L, NaCl 10g/L, yeast extract 5g/L, 2N NaOH to adjust pH up to 7.5

        LB agar plates

        2% agar in LB broth

        Terrific broth

        Tryptone 12g/L, yeast extract 24g/L, glycerol 4ml/L, KH2PO4 3.12g/L, K­2HPO4 12.54g

         

        Buffers:

        Adjust the pH by using 1M KOH solution.

        Buffers used

        Name

        Composition

        pH 6.7 potassium phosphate buffer

        KH2PO4 5.15g/L, K2HPO4 2.1g/L 1M CoCl2 250µl/L

        pH 8.3 potassium phosphate buffer

        KH2PO4 0.5g/L, K2HPO4 8g/L KCl 7.5g/L, 1M CoCl2 250µl/L

        Methyl parathion assay

        200mM CHES buffer 250µl/ml, MilliQ water 750µl/ml, 100µM MPT 1µl/ml

         SDS-PAGE

        12.5% gels were casted for SDS-PAGE for detection of the molecular weight of the proteins.

        Composition of SDS gel

        Name

        12.5 % resolving gel (2 gels)

        4.5% stacking gel (2 gels)

        Acrylamide/bis-acrylamide

        4.1ml

        0.75ml

        1.5M Tris-HCl (pH 8.8) 0.3% SDS

        2.5ml

         

        -

        0.5M Tris-HCl (pH 6.8) 0.4% SDS

         

        -

        1.25ml

        H2O

        3.4ml

        3ml

        10% APS

        100µl

        50µl

        TEMED

        10µl

        5µl

         SDS-PAGE reagents:

        The reagents were prepared and the sample was loaded by adding Laemmli sample buffer and the gel was run in the running buffer. After the electrophoresis, the gel is stained by the staining solution and is further destained by using the destaining solution. 

        SDS-PAGE reagents

         

        Name

        Composition

        Components

        Concentration

        Laemmli sample buffer (2X)

        10% SDS

        Glycerol

        1M Tris-Cl (pH6.8)

        Bromophenol blue

        β-mercaptoethanol

        4%

        20%

        120mM

        0.004%

        10%

        Acrylamide/ bis-acrylamide

        Acrylamide

        N,N’-methylene-bis-acrylamide

        30%

        0.8%

        Running buffer (1X)

        Tris-HCl (pH8.3)

        Glycine

        SDS

        25mM

        200mM

        0.1%

        Staining solution

        Coomassie brilliant blue R250

        Glacial acetic acid

        Methanol:ddH2O (1:1)

        0.25%

         

        10%

        90%

        Destaining solution

        Glacial acetic acid

        Methanol

        ddH2O

        10%

        30%

        60%

        The electrophoresis was carried out at 120V for stacking gel and 200V for resolving gel till the tracking dye gets out of the gel.

        Blue Native PAGE

        The pH of the buffers is adjusted by using HCl. Cathode buffer, divided into three parts, and blue, B/10 and clear buffer are prepared from it.

        Native PAGE reagents

        Name

        Components

        Anode buffer pH 7

        50mM bis tris

        Cathode buffer pH 7

        50mM tricine, 15mM Bis tris

        Blue buffer

        Cathode buffer 200ml, serva blue 0.004

        B/10 buffer

        Cathode buffer 200ml, serva blue 0.0004

        Clear buffer

        Cathode buffer 200ml

        Loading buffer

        Amino cuproic acid 750mM, bis-tris (pH 7) 100mM, serva blue G

        HMW native marker

        (From GE healthcare)

        Pre-casted Native gels were used of thermos fisher scientific (Native PAGE 4-16% Bis-tris protein gels 1.0mm)

        For loading the sample 5X loading dye along with 5µl of glycerol was used.

        The blue native PAGE was performed at 4°C. Initially blue buffer was filled in the tank and ran at 100V for 30 minutes and then at 200V for another 30 minutes, later B/10 buffer was used at 250V for 30 minutes and finally clear buffer was loaded in the tank and the sample was ran at 300V for 40 minutes. 

        Preparation of 0.8% agarose gel

        For preparation of 0.8% agarose gel, agarose was dissolved in 1x TAE buffer and EtBr was added to it at a working concentration of 0.06µg/ml. The agarose gel electrophoresis was performed at 120-150V. The marker used was pure-gene 1kb DNA ladder.

        Components of TAE buffer (10X) are:

        TAE buffer components

        Name

        Amount

        Tris base

        242g

        Acetic acid

        57.1ml

        0.5M EDTA pH8.0

        100ml

        ddH2O

        Up to 1000ml

        Preparation of 6X sample loading buffer

        Sample loading buffer composition

        Name

        Amount

        Bromophenol blue

        5mg

        Xyelene cyanol

        25mg

        Sucrose

        4g

        dd H2O

        Up to 10ml

        The sample loading buffer was stored at 4°C for further use. (Sambrook & Russell, 2001) 

        Western blotting

        The reagents needed for performing the western blot were prepared as per the following protocol.

        The samples were initially electrophoresed over the 12.5% SDS gels and then were transferred to a PVDF membrane. and further the western blotting was performed by using the antibodies to bind to the protein bands.

        The composition of transfer buffer is:

        Composition of Western blot transfer buffer

        Name

        Concentration

        Tris-HCl (pH7.6)

        25m

        Glycine

        192mM

        Methanol

        20%

         The composition of the TBST is:

        Components of TBST buffer

        Name

        Concentration

        Tris-HCl (pH7.4)

        19mM

        NaCl

        137mM

        Tween-20

        0.1%

        Techniques

        Fast protein liquid chromatography.

        In this section we talk about a type of liquid chromatography technique used to analyze and purify the mixture of proteins. Separation occurs on the basis of the interaction of the moving fluid, the mobile phase with the porous matrix being the stationary phase. The flow rate of the buffer is controlled by the pumps and the composition of the buffer can be varied by taking the fluids in different proportions from two different reservoirs.

        FPLC unit consists of two high precision pumps, a control unit, a column, a detection system and a fraction collector. The pumps draw the buffers through a valve and mixing chamber. The flow rate is varied on the basis of the sample to be purified. Injection loops are there to inject the sample into the column. The loop volume varies from 50ul to 50ml. The injection valve links the mixer and the sample loop to the column. The fraction collector is a rotating rack that allows samples to be collected and is controlled by the monitor that shows the peaks of the protein concentration respective to the specific fractions.

        Various columns are used that are packed with the resin and are then filled with the buffer solution. The sample is injected into the column for purification.

        Column chromatography:

        Chromatography is a biophysical technique that is used for fractioning proteins on the basis of the protein charge, shape, size, hydrophobicity and the binding affinity with the stationary phase. The column is filled with a solid matrix and the buffer percolates from it along with the protein samples. The solid matrix is called as the stationary phase while the buffer is known as the mobile phase. Samples are eluted at different flow rates on the basis of the interaction of the sample with the matrix. The matrix has the ions opposite to the charge on the protein and then the protein can be separated by changing the pH or the ionic strength of the buffer or the mobile phase.

        There are various types of column chromatography methods, such as ion exchange chromatography, affinity chromatography, size-exclusion chromatography, high-performnce liquid chromatography and others.

        Ion exchange chromatography

        It is reversible adsorption of charged molecules to the immobilized ion group on the matrix of the opposite charge. Separation is achieved by the adsorption and the release of sample from the matrix. The matrix is associated with the counter ions and when the appropriate conditions for the binding of the sample approach, the sample ions displace the counter ions and bind reversibly to the matrix. The unbound material passes through the matrix and the bound sample is eluted from the column by increasing the ionic strength of the eluting buffer Himmelhoch 1971

        2.png
          Ion exchange column chromatography

          The side (-R) groups of the amino acids are charged and thus the whole of the protein is ionizable or is charged. This property is used to separate different proteins by the ion-exchange chromatography technique.

          On the basis of the matrix bound to it and the type of the protein it will purify, there are two types of ion-exchange chromatography:

          1.    Cation-exchange chromatography

          The matrix of the column is negatively charged and elutes the positively charged cationic proteins, they bind to the matrix and the remaining contaminants come out in the flow through. The proteins with the net positive charge migrate slower as compared to the negatively charged moieties, affected by the interaction of alternatively charged stationary and mobile phase.

          2.    Anion exchange chromatography

          In this technique, the matrix is positively charged and the sample being positively charged interacts with the matrix and thus its flow from the column via the stationary phase is retarded.

          The resolution of the separation of the protein can be enhanced by working along with other properties of the proteins too. As the length of the column increases, the resolution of separation increases. However, the rate of flow of the protein along the column decreases with the increase in the length of the column.

          The resins that are bound along the column are known as ion exchange resins as they are an insoluble matrix with charged groups covalently attached to it.

          There are two types of exchangers:

          1.    Weak exchangers

          Commonly used are carboxymethyl-cellulose (CM-cellulose) and diethylaminoethyl-cellulose (DEAE-cellulose).

          CM-cellulose has carboxymethyl functional group -CH2OCH2COOH. At neutral pH is gets ionized and becomes negatively charged. Thus, it is a weak cation exchanger.

          DEAE-cellulose has diethylaminoethyl group that gets converted to a positively charged group at neutral pH and is thus an anion exchanger.

          2.    Strong exchangers

          Sepharose is used for separation of high molecular weight proteins. Strong exchangers are Q-Sepharose Fast Flow and SP-sepharose Fast flow. The charged group of Q-Sepharose is a quaternary amine and thus carries a non-titratable positive charge. The matrix is used at a alkaline pH.

          The charged group of S-Sepharose is a sulphonyl group (-SO3-). 

          3.png
            Functional groups

             Size-exclusion chromatography​

            This technique also known as molecular sieve chromatography separates proteins on the basis of the size. Large proteins emerge from the column first and then the smaller ones. The matrix consists of beads with pores engineered in them of a particular size. The protein moieties smaller than the pore size enter into the cavities and thus their movement through the column is retarded. The proteins larger in size migrate faster and are eluted first. When an aqueous solution is used to migrate the sample from the column, the technique is known as gel filtration chromatography.

            4.png
              Size-exclusion chromatography

              The advantage of using this technique is that various samples could be eluted without interfering with the fractionation process. It could also be used to determine the approximate molecular weight of the sample by standardizing the flow rate and the compound's molecular weight. An important feature for gel filtration media is that it is inert and thus no sample or compound can bind to it.

              Superdex matrix are generally used for size-exclusion chromatography as it gives high resolution fractionation of the samples. It has a matrix of dextran and agarose cross-linked giving outstanding selectivity and high resolution separation. It is available in three varieties depending on the pore size: superdex 30 prep grade for purification of molecular weight range up to 10000mw, superdex 75 for fractionation range of 3000-70000Da and the superdex200 prep grade for the purification of moieties of the range of 10kDa – 600kDa Boyle 2005

              SDS-PAGE

              In this technique the separation of the macromolecules takes place in the presence of the electric field, known as electrophoresis. The proteins are resolved along the polyacrylamide gel on the basis of their migration rate and SDS is used to denature the proteins. SDS binds to the protein moieties and provides a net negative charge and in the presence of the electric field the protein moieties move towards the anode. It is used to estimate the size of the protein on the basis of the marker used. 

              Blue Native PAGE​

              It is a non-denaturing gel electrophoresis run in the absence of SDS. The mobility depends on both the protein charge and its native size. The electric charge driving the electrophoresis is based upon the intrinsic charge of the protein and the pH of the running buffer used. They are normally carried out at neutral pH in order to avoid the acidic or alkaline denaturation. There are various types of native PAGE, in blue native PAGE CBB provides the charge for the separation of the proteins and even for visualization of the protein bands. In clear native PAGE no dye is used to charge the proteins but it has a lower resolution compared to the BN-PAGE while quantitative native PAGE is a technique used to separate proteins on the basis of their isoelectric point. 

              Constitutive expression of OPH

              The Escherichia coli DH5α cells were earlier cloned for the expression of the OPH synthesizing opd gene under the constitutively expressing lac promoter in pUC19 plasmid. 10 ml of Luria broth (LB) was inoculated with a single colony from the culture plate and incubated overnight at 37°C 180 rpm. 100ml Terrific broth was then inoculated 1% from the LB culture and incubated overnight at 30°C 180rpm. OPH was expressed after 1% inoculation in the terrific broth after 36 hours incubation at 30°C and 180rpm in the presence of the ampicillin. The OPH activity was checked by performing the methyl parathion (MPT) degradation assay.

              The cells were then pelleted down by centrifugation at 6000rpm 4°C for 10 minutes and were washed in pH 6.7 10mM potassium phosphate buffer (PPB). The pellet was then dissolved in 5 times the volume of PPB. The cells were then stirred at 4° along with the addition of 50µg/ml lysozyme for 30 minutes and were later lysed by sonication at 50% amplitude and 20 seconds pulse on and 40 seconds pulse off. The lysate was then centrifuged at 1500rpm and the pellets were discarded.

              The nucleic acid were then precipitated from the supernatant by slowly adding 10% streptomycin sulphate solution prepared in pH6.7 PPB and with continuous stirring at 4°C for 30 minutes. The nucleic acids were discarded by pelleting them by centrifugation at 15000rpm for 30 minutes at 4°C.

              The OPH along with some other proteins were then precipitated by using 45% ammonium sulphate at 4°C and continuous stirring overnight. It was further centrifuged at 15000rpm for 30 minutes at 4°C. The pellet now obtained is then dissolved in the buffer and is then dialysed overnight against the pH6.7 PPB to get rid of the dissolved ammonium sulphate salt. The dialysis buffer was changed after every 4 hours. 

              Purification of OPH

              ​Cation exchange chromatography

              The 30cm SP Sepharose column was packed by dissolving the SP Sepharose beads in milli Q water. The pumps were initially washed with milli Q water and later the pump B was washed with 0.5M KCl solution. The column was washed with Milli Q water and with 0.5M NaOH and then with 1M NaCl. It was then washed with pH 6.7 PPB. The sample was then injected into the FPLC by using the superloop. The flow through was collected and was washed with 2 column volume (CV) of buffer. The OPH along with some contaminants got bound with the column as the pH of the column is below the pI of the OPH and it being positively charged gets bound to the negatively charged SP Sepharose matrix. The OPH was then eluted by using 0.5M KCl solution at the linear gradient of 150ml (5CV). The elution fractions containing OPH was assayed by the MPT degradation assay. The 12.5% SDS gel was casted and the pure protein bands were visualized over it by staining the gel by CBB and further destaining using the destain solution.

              The fractions having the OPH were then collected and dialysed against the pH 8.3 PPB. The dialysis was kept overnight and buffer was then changed and kept for 4 hours. 

              Anion exchange chromatography

              The 20cm DEAE Sepharose column was packed by dissolving the matrix beads in the milli Q water. Later the column was washed by using 0.5M NaOH and then with1M NaCl followed by pH 8.3 PPB. The dialysed sample was then injected into the column. The pH of the column was 8.3 and the matrix DEAE sepharose is positively charged. The proteins having negative charge gets bound to the column. OPH having pI of 8.4 thus at pH 8.3 it is neutral or slightly positively charged and was then obtained in the flow through only.

              The fractions were then tested by the MPT degradation assay and in order to confirm 12.5% SDS gel was casted and SDS-PAGE was performed. The protein sample were concentrated to 2ml by using Amicon ultra-15 10K centrifugal filter device by centrifugation at 3500rpm 4°C. 

              ​Size-exclusion chromatography​

              The 60cm column was packed by using Superdex 200 prep grade matrix dissolved in milli Q water. The column was then washed using milli Q water initially and then with 50mM HEPES buffer pH 8.3. The protein sample was then injected into the FPLC column. Superdex 200 prep grade has fractionation size of 10kDa – 600kDa. OPH is being purified from the contaminants on the basis of the flow rate based upon the size of the proteins.

              The fractions collected were checked for the MPT degradation assay and they consist of purified OPH was assured by running the elutions over the SDS-PAGE.

              Protein concentration assay

              The concentration of the purified OPH was determined by performing the bicinchoninic assay and absorbance measured at 562nm. 

              Determination of molecular mass in native condition

              To determine the molecular mass of the OPH in its native conditions, Blue native PAGE was performed of the OPH obtained after the purification from the above techniques.

              Cloning of His-tag in opd

              The pUC19 with wt opd and pUC19 with mutant opd plasmid harbouring E.coli cells were inoculated in 10ml LB culture and then the plasmids were isolated using the thermos fisher plasmid isolation kit method. 0.8% agarose gel was casted and plasmid isolation was verified by the bands over the gel. The concentration of the plasmids was measured using Nanodrop.  

              Restriction digestion

              opd gene has SalI site at 515bp structure in it and BamHI site was available in the multiple cloning region of the plasmid. Further 1µg of plasmid was digested by using SalI and BamH1 restriction enzymes in order to get a release of the C-terminal of the OPH and the digested plasmid was extracted from the agarose gel by the Nucleopore gel extraction kit.

              The restriction digestion was kept in a reaction of 20µl and was digested in steps, initially by one enzyme and later the linearized plasmid was digestion with the other RE to get the fragment released of the plasmid. Fast digest enzymes were used for the digestion reaction.

              Template 1µg

              10X fast digest buffer 2µl

              Restriction enzyme 1µl/µl

              Milli Q water upto 20µl

              The reaction mixture was incubated for 1hour at 37°C.

              Restriction digestion of pPHNS

              pPHNS was available in the form of glycerol stock in the lab cloned in pET23b and in BL21 cells. The 10ml LB was inoculated from the glycerol stock and the plasmid was isolated using the plasmid isolation kit protocol. The BglII restriction site was engineered in the pPHNS plasmid to digest the plasmid and to clone it in the pUC19 plasmids later.

              The PCR was done by using T7 BglII forward and reverse primers.

              The PCR was kept at the given program.

              Initial denaturation 95°C 5minutes

              Denaturation 95°C 45seconds

              Annealing 56°C 45seconds 30cycles

              Elongation 72°C 1minute 30 seconds

              Final elongation 72°C 5minutes

              Storage 12°C inifinite

              The SalI site was there in the opd gene and by the use of T7 BglII primers a BglII site is inserted in the plasmid. BglII RE site shows homology with the BamHIsite thus the cells by their repair mechanism can ligate BamH1 with the BglII site.

              The PCR product was purified by using the nucleopore PCR clean-up kit.

              Then the PCR product was then digested using the BglII and SalI RE using the sample protocol used earlier. The digested fragment was extracted and then purified using the nucleopore gel extraction kit. The concentration of the digested product was measured using the nanodrop. 

              Alkaline phosphatase treatment of Vector

              The pUC19 vector was treated with alkaline phosphatase (AP) in order to minimize the chances of the ligation in the reverse orientation. The reaction was set up as 20µl reaction. The reaction was incubated at 37°C for 10 minutes and AP was then heat inactivated by incubation at 75°C for 5 minutes. 

              Ligation of SalI BglII fragment inpUC19

              The ligation was performed as per the neb ligation calculator. The 100ng vector reaction was set up and according to it the insert was added in 1:3 and 1:5 ratios on the basis of their concentration.

               Required mass insert (ng) = Mass of vector (ng) * [size of insert(kbp) / Size of vector (kbp) * insert: vector molar ratio]

              Controls were also prepared in order to check the self-ligation capability of the the vector. The 20µl reactions were kept and were incubated at 4°C for 12 hours.

              Competent cell preparation

              10ml of LB was inoculated by a single DH­5α colony and was incubated overnight at 37°C and 180rpm. Another 10ml LB was inoculated (100µl) from the primary culture and incubated to upto the log phase of the cells at 37°C and 180rpm. The culture was taken at an OD of 0.5 and then 1ml culture was taken up in different microcentrifuge tubes and was centrifuged at 4°C for 5minutes at 6000rpm. To the pellet obtained, 1ml ice cold 0.1M CaCl2 was added and pellet was dissolved in it. The sample was kept on ice for 30 minutes. The culture was again centrifugated at 4°C for 5minutes and at 6000rpm. The pellet obtained is now dissolved in 100µl of 0.1M CaCl2 and were used for the transformation. 

              Transformation of ligation reaction to the competent cells

              The ligation reaction was added to the competent cells and was kept on ice for 30minutes. After that a heat shock was given to the sample for 90seconds at 42°C. After that 1ml LB was added to the sample and was kept for incubation at 37°C 180rpm for 1 hour. The culture was then pelleted by centrifugation at 6000rpm for 5 minutes. The pellet was dissolved in 50µl of LB and was spread over the LB+ Ampr plates. The plates were incubated at 37°C for 16hours 

              Screening of the transformants

              The transformed colonies were screened by performing colony PCR by using M13 forward and reverse primers. The colonies were inoculated in 20µl of milli Q water and were then kept in boiling water for 10 minutes in order to lyse the cells. The 20µl PCR reaction was kept for the colonies at the given program.

              Initial denaturation 95°C 5minutes

              Denaturation 95°C 45seconds

              Annealing 54°C 45seconds 30cycles

              Elongation 72°C 1minute 30 seconds

              Final elongation 72°C 5minutes

              Storage 12°C inifinite

              0.8% agarose gel was casted and the colony PCR products were screened on the gel.

              The colonies giving the desired size fragments were inoculated in 10ml LB+ Ampr culture and were incubated overnight at 37°C 180rpm. The plasmid was isolated from these colonies and was then checked for the digestion with Xho1 RE. Xho1 is the restriction site that is not present in pUC19 opd plasmid while it is present in the pET23b cloned opd as the opd was cloned in pET23b between Nde1 and Xho1 sites. So, if the pET23b fragment has been cloned in pUC19+opd plasmid then it will be showing linearization by the Xho1 RE. The colonies linearized by the Xho1 enzyme were validating that the cloning has been done.

              Western cloning

              The cloned colonies 0.5ml culture was pelleted and was then dissolved in 100ul of 2X sample loading dye. The sample was then kept in boiling water for 10minutes to lyse the cells.

              12.5% SDS gels were casted and the saples were then resolved over the SDS-PAGE. The bands were transferred to the PVDF membrane by applying the electric field. The PVDF membrane was then incubated for 1 hour with the blocking buffer (5% skimmed milk in TBST buffer) to minimize the undesired binding of the priary antibody to the membrane. After the blocking 3 washings were done of the PVDF membrane with the TBST buffer, 5minutes each in order to remove the unbound blocking buffer. Further 1ul/10ml anti-His tag antibody was prepared in skimmed milk. The membrane was incubated with the antibody for 1 hour and after that 3 TBST washing were given for 5 minutes each.

              The blot was then developed by using Biorad luminol and peroxidase as the substrate.

              Results

              Various chromatographic techniques were used to purify the OPH. The pure OPH was around 35kDa in its monomeric form and in its native form it is observed as 150kDa.

              Purification by cation exchange column chromatography

              Purification was performed by using SP Sepharose matrix column. Negatively charged contaminants were initially eliminated in the flow through while the positively charged OPH was obtained in the elution at 125mM KCl solution with an absorbance peak of 50mAU.

              5.png
                FPLC cation exchange column chromatography

                When the sample was resolved on the SDS-PAGE, the pure OPH gave a 37kDa band and very few contaminants were observed on the gel. The fractions were then obtained and used for anion exchange column chromatography.

                6.jpg
                  SDS-PAGE of cation exchange column chromatography purified OPH

                  Purification by Anion exchange column chromatography:

                  The pure OPH was obtained on the flow through only while purifying through the DEAE sepharose packed column. The OPH gave a peak of 25mAU and was obtained as a single peak without any contaminants.

                  7.png
                    FPLC anion exchange column chromatography

                    The purified OPH was resolved on the 12.5% SDS-PAGE and 357kDa band was obtained and the fractions were then collected and were processed for further purification by gel filtration chromatography.

                    8.jpg
                      SDS-PAGE of anion exchange chromatography purified OPH

                      Size-exclusion column chromatography

                      Gel filtration was performed using superadex 200 prep grade bead matrix. The large size protein were initially obtained in very small fractions and later at around 100ml of the buffer the pure OPH was obtained giving a absorbance of 50mAU.

                      9.png
                        FPLC size-exclusion column chromatography

                        The fractions obtained were then run over the SDS-PAGE and purified OPH bands of 35kDa were observed in the fractions. It was observed that the OPH fractions were there without any contaminants.

                        10.jpg
                           SDS-PAGE of size-exclusion chromatography purified OPH

                          Estimation of concentration of OPH:

                          The concentration of the purified OPH was 5mg/ml as estimated by BCA protein estimation assay.

                          Blue- Native PAGE

                          The purified OPH obtained from the gel filtration technique were than resolved on the Native gel and a band of apporimatly150kDa was observed on the native PAGE.

                          This depicts that the monomeric form of the OPH is of 35kDa and in the native state the OPH is of around approximately 150kDa. This shows that the OPH is tetramer in its native conditions.

                          12.jpg
                            Native-PAGE of OPH

                            This depicts that the monomeric form of the OPH is of 35kDa and in the native state the OPH is of around approximately 150kDa. this shows that the OPH is tetramer in its native conditions.

                            Cloning of His-tag from pPHNS to pUC19

                            The pUC19+opd plasmid is 3.7kb in size and on digesting the plasmid with SalI and BamHI, a 900bp release was obtained in the agarose gel. Another band of 3kb was extracted from the agarose gel.

                            puc19.JPG
                              pUC19 digested with SalI and BamHI

                              pPHNS plasmid was isolated and then the opd fragment was amplified by performing PCR using M13 forward and reverse primer. A 3' band of 1.2kb was obtained after the PCR. It was then purified using PCR clean-up kit. It was then digested with SalI and BglII. A 5' 900bp band was obtained after the digestion and was extracted from the agarose gel.

                              pPHNS.JPG
                                pPHNS digested with SalI and BglII

                                The concentrations of the insert and vector were:

                                Name

                                Concentration (ng/µl)

                                pUC19+wt opd vector digested with SalI and BamH1

                                22.88

                                pUC19+mut opd vector digested with SalI and BamH1

                                25.13

                                pPHNS insert digested with SalI and BglII

                                29.2

                                 

                                The ligation was kept according to these concentrations.

                                cPCR.JPG
                                  cPCR of the transformants

                                  linearized.JPG
                                    XhoI digestion of Transformants

                                    The transformants obtained were verified to be clones after performing colony PCR by using M13 forward and reverse primers. The clones giving the band of 1.4kb were selected as to be clones and plasmid was isolated from them. After digesting with Xho1 a linearized band of 4kb was observed on the agarose gels. This linearized plasmid band validates that the clones has been obtained.

                                     Western Blot of OPH

                                    The western blot was developed by using luminol and peroxidase as the substrate. A band of around 35kDa was obtained by using anti-His tag antibodies for the blot and this confirms that the vector has been cloned by the His tag along with the OPH and is getting expressed too.

                                    Capture.JPG
                                      Western blot ofpUC19 opd + His tag

                                      Thus, these clones were identified as colonies having pUC19 having opd+His tag gene in them.

                                      Conclusion

                                      It could be said that the organophosphate hydrolase exists as a tetramer in its native state because when the size of the purified protein was checked on the native gel it was giving the band of 150kDa and on the SDS-PAGE the purified form of the protein was giving the band of 37kDa. However, the results are needed to be validated by performing MALDI-TOF mass spectroscopy and further we need to confirm the molecular structure of the OPH by cryo-electro microscopy. For fastening the process of the purification of the OPH, we cloned the His-tag in the opd gene in the pUC19 vector and confirmed its expression. Thus, by constitutively expressing the OPH in the pUC19 plasmid, we can easily purify the protein by using the Ni-NTA purification protocol by using the His-tag of the OPH.

                                      Acknowledgements

                                       I would like to take this opportunity to thank Indian Academy of Sciences for providing me the opportunity to work under the Summer Research fellowship programme in the University of Hyderabad. I extend my heartful gratitude to Prof. Siddavattam Dayananda, for providing me the opportunity to work under his excellent supervision. His vital support and valuable suggestions have played a big role in shaping this project work. I would like to express my sincere thanks to my mentor Mr. Hari Parapatla for always being there to guide me with his knowledge and motivation and helping me to learn an array of techniques in this duration. I am very grateful for his boundless efforts and supervision. I am also grateful to Ms. Harshita Yakkala, Ms. Annapoorni Lakshman Sangar, Ms. Ganeshwari Dhruve and Mrs. Devyani Samantani for helping me to know the basics of molecular biology laboratory to carry out my project work and creating the pleasant atmosphere of work in the laboratory. I would also like to thank Mr. Ramamurthy Gudla, Dr. Rajesh, Mr. Shaikh Akbarpasha, Ms. Aparna and lab assistant Mr. Krishna for their timely help and suggestions. I would also like to thank my friends in the university and especially, Ms. Baby Kumari for providing me the family atmosphere. Finally, I express my deep gratitude and appreciation to almighty, my parents and my sisters for their support and enormous encouragement.

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                                      • Sunil Parthasarathy, Hari Parapatla, Dayananda Siddavattam, 2017, Topological analysis of the lipoprotein organophosphate hydrolase from Sphingopyxis wildii reveals a periplasmic localisation, FEMS Microbiology Letters, vol. 364, no. 19

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