Bioseparation9: 155–161, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Purification of alkaline phosphatase from chicken intestine by three-phasepartitioning and use of phenyl-Sepharose 6B in the batch mode

Aparna Sharma, Shweta Sharma & M.N. Gupta∗Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi-110 016, India

Received 20 January 2000; accepted in revised form 14 June 2000

Key words: chicken intestine alkaline phosphatase, phenyl Sepharose, three-phase partitioning

Abstract

Alkaline phosphatase from chicken intestine was purified from the crude preparation employing three-phase parti-tioning and by the use of phenyl Sepharose-6B in the batch mode. TPP uses a combination of ammonium sulphateand t-butanol to precipitate proteins from crude aqueous extracts. The precipitated protein forms interface betweenlower aqueous phase and upper organic solvent phase. The fold purification of the finally purified enzyme was 80and the activity recovery was 61%. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis ofenzyme showed considerable purification and its molecular weight was found to be around 67 kDa.

Abbreviations:TPP – three-phase partitioning, AP – alkaline phosphatase, SDS-PAGE – sodium dodecyl sulphate-polyacrylamide gel electrophoresis, BSA – bovine serum albumin

Introduction

Alkaline phosphatase (E.C.3.1.3.1) is commonly usedas a label and signal amplifier in enzyme-linked im-munosorbent assay [ELISA] (Sanden et al., 1998,Unson et al., 1999), for analytical and preparativeapplications in molecular biology research and for de-phosphorylation of phosphoproteins and nucleotides(Pappas et al., 1998, Kassner et al., 1999). Two mainsources for obtaining the enzyme for above applica-tions areE. coli (Basheeruddin et al., 1985) and calfintestine (Kirchberger and Kopperschlager, 1982). Inboth cases, the purification protocols described aremultistep and time consuming. In recent years, thetrend has been to develop efficient, economical andscaleable approaches for bioseparation of enzymesand proteins (Gupta and Mattiasson, 1994; Sharma etal., 2000). This is particularly relevant for enzymeslike alkaline phosphatase which are required for spe-cific applications and in large amounts. Recently, wehave described purification of alkaline phosphatasefrom chicken intestine on Cibacron Blue dye-linkedcellulose beads. The one step protocol consisting ofexpanded bed chromatography yielded an enzyme

with 48 fold purification with an activity recovery of70%. However, the purified enzyme was still hetero-genous with at least four bands on SDS-PAGE. In thiswork, we describe an alternative two step approachwhich yields an enzyme preparation of higher purityand comparable yield.

TPP is a relatively recent and underexploitedtechnique for the protein purification (Dennison andLovrein, 1997). Tertiary butanol is a water misciblesolvent but its aqueous solution separates into twophases when enough salt is added. When an aqueousextract of protein solution containing adequate amountof salt is mixed with t-butanol, then the protein mayform an interfacial precipitate separating the loweraqueous phase and upper t-butanol phase. Thus, thetechnique uses the combination of salt and t-butanolto precipitate proteins from crude aqueous extracts.The proteins/enzymes have been observed to retaintheir biological activities in t-butanol–water mixtures.Hence, such three-phase partitioning (TPP) can beused to isolate and concentrate proteins. In fact, usu-ally there is a window of optimal range of salt in TPPaqueous phase below which the desired enzyme willnot form interfacial precipitate and above which other

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contaminating proteins will also become part of thisprecipitate. Thus, by using range of salt concentration,one can bring in selectivity and use TPP for proteinpurification as well. Generally, ammonium sulphatehas been used as salt. Other organic solvents, apartfrom t-butanol have also been tried but t-butanol hasbeen found to work best in most of the cases so far.Quite a few successful applications of this approachhave been described (Dennison and Lovrein, 1997).In the present purification scheme, three-phase parti-tioning followed by hydrophobic interaction chroma-tography gave a highly purified enzyme preparation.

Materials and methods

Chicken intestine was procured from the local mar-ket. p-Nitrophenylphosphate was purchased fromSisco Research Laboratories, Mumbai, India. Phenyl-Sepharose was purchased from Sigma Chemical Co.,St. Louis, MO, USA. All solvents used were of HPLCgrade and all other reagents were of analytical grade.

Enzyme activity and protein assay

Alkaline phosphatase activity was measured accordingto the method described by Maunders (1993) with aslight modification. The assay buffer consisted of 1 Mdiethanolamine containing 0.0005 M MgCl2at pH 9.8.One unit of enzyme activity is defined as the amountof enzyme which releases 1µmol of the product (p-nitrophenol) per minute under the assay conditions.

Protein concentration was determined by the dyebinding assay (Bradford, 1976) using BSA as thestandard.

Isolation of alkaline phosphatase from chickenintestine

The crude extract of alkaline phosphatase was pre-pared according to Chang and Moog (1972). Thechicken intestinal loop (25 g) was washed thoroughlyto remove attached fat, etc. Each loop was homogen-ized in a blender and 165 ml distilled water was addedto the whole tissue content followed by addition of 165ml n-butanol. Then mixture was stirred for 1 hour at4 ◦C followed by stirring at 40◦C for 10 min. Theextract was then centrifuged at 12,000 g for 20 minand two layers (aqueous and organic solvent) wereformed. The aqueous layer containing enzyme activitywas collected and dialyzed against distilled water for72 h. The dialyzed extract was centrifuged to remove

any remaining tissue particles which was then used ascrude preparation for subsequent studies.

Purification of alkaline phosphatase by three phasepartitioning (TPP) in aqueous / organic cosolventmixturesTPP was carried out by saturating the enzyme solu-tion (2000 U) with 30% ammonium sulfate (w/v)and adding various organic solvents (t-butanol, 1,4-dioxane, tetrahydrofuran, isopropanol, n-propanol, n-amylalcohol, t-amylalcohol, dimethylsulphoxide, di-methylformamide and formamide) in the ratio of 1:1(v/v) (ratio of crude extract to organic solvent). Thesolutions were then kept at 25◦C for 2 h for completephase separation. The mixtures were then centrifugedat 2000g for 10 min and the three phases formedwere collected separately. Since the enzyme was notprecipitated completely in the middle-layer in the firststage, the lower aqueous layer was used to carry outthe second stage of partitioning. The aqueous layer ob-tained in the first stage was again saturated with 30%ammonium sulphate (w/v) (such that final concentra-tion was 60% (w/v) followed by addition of an equalvolume of the organic solvent (Pike and Dennison,1989).

The solutions were again incubated for two hoursat 25 ◦C followed by centrifugation at 2000g for10 min. The interfacial precipitate was collected andenzyme activity was determined in the precipitate.

Hydrophobic Interaction Chromatography (HIC) ofthe partially purified enzyme

Phenyl-Sepharose (2.0 mL) was washed with distilledwater and then pre-equilibrated by washing with 0.05M Tris-HCl, pH 7.0 buffer containing 1 M ammoniumsulfate. The precipitate was equilibrated using 0.05 MTris-HCl buffer, pH 7.0 containing 1.0 M ammoniumsulfate. 1.0 mL of this precipitate (1771 U) was addedto the matrix (after draining off excess buffer) and fi-nal volume was made to 4.0 mL with the equilibratingbuffer. The mixture was kept at 30◦C for 1 h undershaking conditions for binding and then centrifuged at12,000g for 10 min. Further batch washings of theresidue were carried out in the same buffer till therewas no detectable enzyme activity in the supernatant.Desorption of the bound enzyme activity was carriedout using 2.5 mL of 50% ethylene glycol in 0.05 MTris-HCl buffer, pH 7.0 for 3 h at 10◦C. After centri-fugation, the desorbed activity was determined in thesupernatant.

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Table 1a.Purification of alkaline phosphatase by TPP method from crude chicken in-testine extract in presence of different organic solvents. Organic solvents (2.0 mL) wereadded to 2.0 mL of AP crude chicken intestine extract (2000 U) saturated with 30% (w/v)ammonium sulfate. Recovery of target enzyme was carried out as described in the ‘Mater-ials and methods’ section. Other solvents tried were n-amylalcohol, dimethylformamide,dimethylsulphoxide and formamide. In all these solvents, the formation of the third phasewas not observed even when 2.0 mL of the crude extract was mixed with 6.0 mL of theorganic solvent

Organic Activity Protein Specific Fold Activity

solvents (Units) (µg) activity purification yield (%)

(Units mg−1)

Crude 2000 2400 834 1 100

t-butanol 1771 94 18840 23 88

t-amyl alcohol 1107 65 17038 20 55

1,4 Dioxane 1400 134 10447 13 70

Tetrahydrofuran 826 32 27533 33 41

Isopropanol 508 164 793 1 13

n-propanol 1250 204 6127 7 62

Table 1b. Distribution of protein in the TPP system. Organic solvents (2.0 mL) wereadded to 2.0 mL of alkaline phosphatase crude extract from chicken intestine (2000U) saturated with 30% (w/v) ammonium sulfate. Rest of the procedure was same asin ‘Materials and methods’ section. Protein was estimated in all the three layers afterthe first and the second phase of TPP. The organic solvent layer did not contain anysignificant amount of protein. The total amount of protein present in crude was 2400µgin each case

Protein (µg)

Organic Precipitate 1a Aqueous 1b Precipitate 2c Aqueous 2d

solvents

t-butanol 700 1700 94 1560

t-amyl alcohol 300 1800 65 1760

1,4 Dioxane 569 1600 134 1490

Tetrahydrofuran 463 1800 32 1650

Isopropanol 520 1652 164 1488

n-propanol 320 1600 204 1480

aPrecipitate 1: The interfacial precipitate obtained after first TPPbAqueous 1: The aqueous layer obtained after first TPPcPrecipitate 2: The interfacial precipitate obtained after second TPPdAqueous 2: The aqueous layer obtained after second TPP

SDS-Polyacrylamide gel electrophoresis

SDS-PAGE of the purified enzyme with 10% gelstrength was performed according to Laemmli (1970)using BioRad Mini Protean II electrophoresis unit.

Results and discussion

Three-phase partitioning employs collective operationof principles involved in numerous techniques such

as conventional salting out, Morton’s n-butanol ex-traction method, isoionic precipitation, cold cosolventprecipitation and osmolyte and kosmotropic precip-itation of proteins. The technique promises severaladvantages. It is easily scaleable and can be useddirectly with crude suspensions. It is a concentrat-ing or dewatering step and concentration factors up to100X have been reported (Lovrien et al., 1995). AsTPP requires less salt than in conventional ‘salting in’approach but requires additional expenditure on or-

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Table 2a. Optimisation of the binding conditions for alkaline phosphatase to phenylsepharose beads. 1.0 mL of the partially purified enzyme was added to 2.0 mL of preequilibrated phenyl sepharose beads and the final volume was made to 4.0 mL withthe equilibrating buffer. Rest of the procedure was same as described in ‘Materials andmethods’ section. Bound activity is expressed by taking the starting activity (after TPP)as 100%

Binding conditions % Activity

bound

0.05 M Tris-HCl, pH 7.0, 30◦C, 1 h 54

0.05 M Tris-HCl, pH 7.0, containing 0.5 M (NH4)2SO4, at 30◦C, 1 h 78

0.05 M Tris-HCl, pH 7.0, containing 1.0 M (NH4)2SO4, at 30◦C, 1 h 98

Table 2b. Optimization of desorption conditions for alkaline phos-phatase bound to phenyl sepharose beads. 1.0 mL of the partiallypurified enzyme was added to 2.0 mL of pre equilibrated phenylsepharose beads and the final volume was made to 4.0 mL withthe equilibrating buffer. The mixture was then kept at 30◦C for1h under shaking conditions for maximal binding of the enzyme.Washings were carried out till there was no detectable activity inthe supernatant. 2.5 mL of the eluants (ethylene glycol of variedpercentage was made in 0.05 M Tris-HCl, pH 7.0) were addedto desorb the bound enzyme. Rest of the procedure was same asdescribed in ‘Materials and methods’ section. Desorbed activity isexpressed by taking the total bound activity as 100%

Elution % Enzyme Fold

recovery purification

Tris-HCl buffer, 10◦C, 3 h 23 1.7

50% ethylene glycol, 10◦C, 3 h 68 3.6

50% ethylene glycol, 10◦C, 24 h 74 1.0

80% ethylene glycol, 10◦C, 3 h 70 1.2

ganic solvent, the cost is marginally higher (than insalt precipitation). However, fold purification is gener-ally found to be much higher (Dennison and Lovrien,1997).

Table 1a shows the results of three phase par-titioning with crude extract of chicken intestine al-kaline phosphatase using various solvents. Of all thesolvents tried t-butanol gave the best results with 23fold purification and 88% activity recovery. This isin agreement with the experience of others (Dennisonand Lovrien, 1997). It is believed that because of itssize and branched structure, t-butanol does not eas-ily permeate inside the folded protein molecules andhence does not cause denaturation (Lovrein et al.,1987). In the present system, although tetrahydrofurangave higher fold purification, the activity recoverywas much lower. Table 1b gives the protein presentin aqueous phase and in the precipitate at various

stages. It appears that both higher deactivation of theenzyme as well as lower interfacial precipitate are re-sponsible for this lower activity yield in the case oftetrahydrofuran.

Figure 1 shows the effect of varying ammoniumsulfate concentration on the recovery of enzyme activ-ity and protein in the interfacial precipitate phase. Itwas seen that 30% ammonium sulfate gave best res-ults. Further addition of higher ammonium sulfategave direct protein precipitation indicating that theconditions for conventional salt precipitation havebeen reached. Efforts were also made to optimize theratio of the volume of organic solvent to the volume ofaqueous crude extracts. The best results were obtainedwhen the ratio was 1:1 (data not shown). Thus the op-timum conditions for TPP were the use of t-butanolalong with 30% ammonium sulfate and 1:1 ratio oft-butanol to aqueous crude extract.

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Table 3a. Purification table: Purification of alkaline phosphatase from crude extract bythree-phase partitioning using t-butanol and hydrophobic interaction chromatographyusing phenyl sepharose beads. After TPP step, the interfacial precipitate was dialysedagainst 0.05 M Tris-HCl buffer, pH 7.0 containing 1 M for pre- equilibration prior tobinding with phenyl sepharose beads. Rest of the procedure was same as described in‘Materials and methods’ section

Steps Activity Protein Specific Fold Activity

(Units) (µg) activity Purification Yield

(Units mg−1) (%)

Crude 2000 2400 834 1 100

TPP 1771 94 18840 23 88

Phenyl sepharose 1210 18 67222 80 61

beads

Table 3b. Purification table:Purification of alkaline phosphatase from crude extract by am-monium sulphate (70%) precipitation and hydrophobic interaction chromatography usingphenyl sepharose beads After ammonium sulphate step, the interfacial precipitate was dis-solved in 0.05 M Tris-HCl buffer, pH 7.0 containing 1 M ammonium sulphate prior to bindingwith phenyl sepharose beads. Rest of the procedure was same as described in ‘Materials andmethods’ section

Steps Activity Protein Specific Fold Activity

(Units) (µg) activity Purification Yield

(Units mg−1) (%)

Crude 1080 1500 720 1 100

Ammonium sulphate 864 171 4932 7 80

precipitation

Phenyl Sepharose 572 42 13619 19 53

beads

Table 3c. Purification table: Purification of alkaline phosphatase from crude extract bythree- phase partitioning using t-butanol and hydrophobic interaction chromatographyusing phenyl sepharose beads. After TPP step the dialysis step was omitted, the interfacialprecipitate was dissolved in the equilibrating buffer (0.05 M Tris-HCl, pH 7.0 containing1 M ammonium sulphate) prior to binding with phenyl sepharose beads. Rest of theprocedure was same as described in ‘Materials and methods’ section

Steps Activity Protein Specific Fold Activity

(Units) (µg) activity Purification Yield

(Units mg−1) (%)

Crude 1080 1500 720 1 100

TPP 939 55 17073 24 87

Phenyl sepharose 726 12 59040 82 68

beads

The analysis of the enzyme preparation obtainedafter TPP on SDS-PAGE showed multiple bands (Fig-ure 2). At least three bands could be clearly seen.

We had earlier observed that the intestinal alkalinephosphatase binds to a hydrophobic matrix (Agarwaland Gupta, 1995). Hence, the enzyme preparation ob-tained after TPP was purified on a phenyl-Sepharose

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Figure 1. Effect of ammonium sulphate concentration on the crude intestinal extract in presence of tertiary butanol. To 2.0 mL of the crudechicken intestine extract (containing 2000 U) was added different amount of ammonium sulphate (10%, 20%, 30% w/v−1) keeping the volumeof t-butanol constant (2.0 mL). The three phases formed (after second stage of precipitation) were collected separately. Recovery of targetenzyme act ivity ( ) and protein ( ) was done as described in ‘Materials and method’

Figure 2. SDS-PAGE pattern of purified alkaline phosphatase D. Lane 1: crude alkaline phosphatase; Lane 2: marker proteins; Lane 3: purifiedalkaline phosphatase; Lane 4: partially purified alkaline phosphatase after TPP.

matrix in the batch mode. The enzyme activity couldbe bound almost quantitatively (98%) in the pres-ence of 1 M ammonium sulphate (Table 2a). About68% of the enzyme activity could be desorbed with50% ethylene glycol in 0.05 M Tris-HCl buffer, pH7.0 (Table 2b) with 3.6 fold purification. Finally theenzyme was 80 fold purified (Table 3a). This SDS-PAGE showed a considerable purification (Figure 2).The molecular weight of nearly 67 kDa is similar tothe reported values of 69 kDa for the monomer of

calf intestine alkaline phosphatase (Kirchberger andKopperschlager, 1982).

It may be interesting to compare this TPP basedstrategy with the one based upon conventional salt pre-cipitation. It was found that 60% ammonium sulphate(w/v) did not precipitate any protein/enzyme. Table 3bshows the results obtained of precipitation with 70%(w/v) ammonium sulphate followed by hydrophobicinteraction chromatography step. While activity yieldwas only slightly less, fold purification was merely19 as compared to 80 obtained with TPP based

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strategy (Table 3a). This difference in fold purifica-tion is largely at the step of using salt precipitation.While conventional ammonium sulphate precipitationgave only 7 fold purification. TPP with much lowersalt concentration gave 23 fold purification. Table 3cshows the results obtained if dialysis is omitted afterTPP step and before proceeding to HIC step. Omittingthis dialysis step did not affect the fold purification andyield, infact, was marginally better. Thus the dialysisstep can be safely omitted which makes the approachmore conveniently scalable. All the experiments weredone in duplicate and the difference in the results induplicates was less than± 5%

The above data, apart from describing a two stepefficient protocol for purification of an alkaline phos-phatase also shows the power of TPP as an initial stepfor bioseparation of proteins/enzymes.

Acknowledgements

This work was partially supported by project fundsfrom Council for Scientific and Industrial Research(CSIR) and Department of Biotechnology (Govt. ofIndia organisation). The financial support provided byIndian Institute of Technology, Delhi in the form of Ju-nior Research Fellowship to SS is also acknowledged.

References

Agarwal R and Gupta MN (1995) Evaluation of glutaraldehyde-modified chitosan as a matrix for hydrophobic interaction chro-matography. Anal. Chim. Acta. 313: 253–257.

Basheeruddin K, Rothman V and Margolis S (1985) ImmobilizedE. coli alkaline phosphatase – Its properties, stability and utilityin studying the dephosphorylation of proteins. Appl. Biochem.Biotechnol. 11: 133–140.

Bradford MM (1976) A rapid and sensitive method for the quantita-tion of microgram quantities of protein utilizing the principle ofprotein-dye binding. Anal. Biochem. 72: 248–254.

Chang CH and Moog F (1972) Alkaline phosphatase of the chickenduodenum I. Isolation and partial characterization of the multipleforms of duodenal phosphatase in pre- and post hatching stages.Biochim. Biophys. Acta 258: 154–165.

Dennison C and Lovrein R (1997) Three-Phase Partitioning: con-centration and purification of proteins. Protein Expr. Purif. 11:149–161.

Kassner AM, Lessmann M and Wasner H (1999) Degradation of thecyclic AMP antagonist prostaglandylinositol cyclic phosphate(cyclic PIP) by dephosphorylation. Biol. Chem. 380: 85–88.

Kirchberger J and Kopperschlager G (1982) Preparation of homo-genous alkaline phosphatase from calf intestine by dye-ligandchromatography. Prep.Biochem. 12: 29–47.

Maunders MJ (1993) Alkaline Phosphatase (EC 3.1.3.1): "Enzymesof Molecular Biology". Burrell MM, (Ed.) pp 331–341, HumanaPress, New Jersey.

Gupta MN and Mattiasson B (1994) Novel technologies in down-stream processing. Chem. & Indus., 673–675.

Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227: 680–685.

Lovrein RE, Goldensoph C, Anderson P and Odegard B (1987)Three Phase Partioning (TPP) via t-butanol: enzyme separationfrom crudes In: Burgess, R. (Ed.), "Protein Purification: Microto macro." p 131–148. Alan R. Liss, Inc., New York.

Lovrien RE, Conroy MJ and Richardson TI (1995) Molecular basisfor protein separations. In: Gregory, R.B., (Ed.), "Protein-solventinteractions" pp 521–553. Marcel Dekker Inc., New York

Pappas A, Yang WL, Park TS and Carman GM (1998) Nucleotide–dependent tetramerization of CTP synthetase fromSaccharomy-ces cerevisiae.J. Biol. Chem. 273: 15954 –15960.

Pike RN and Dennison C (1989) Protein fractionation by three-phase partitioning in aqueous/ t-butanol mixtures. Biotechnol.Bioeng. 33: 221–228.

Sanden B, Eng LH and Dalhammar G (1998) An amperometricenzyme–linked immunosensor for nitrobacter. Appl. Microbiol.Biotechnol. 50: 710–716.

Sharma A, Sharma S and Gupta MN (2000) Purification of wheatgerm amylase by affinity precipitation. Protein Expr. Purif. 18:111–114.

Unson MD, Newton GL, Arnold KF, Davis CE and Fahey RC(1999) Improved methods for immunoassay of mycothiol. J.Clin. Microbiol. 37: 2153–2157.