application of vesicle chromatography in protein purification

Acta Biotechnol. 12 (1992) 3, 243-253 Akademie Verlag

Application of Vesicle Chromatography in Protein Purification

KLEINE, R. ', WOEHLECKE, H. *, EHWALD, R.

Technische Hochschule Kiithen, Institut f i r Biologie und Biochemie Bernburger Str. 52-57,04370 Kiithen, F.R.G.

Fachbereich Biologie Invalidenstr. 42, 0-1040 Berlin, F.R.G.

* Humboldt-UniversitHt N Berlin

Summary

A recently described vesicular packing material (VP) representing clusters of microcapsules (derived from plant cells) was tested with respect to its application for protein purification. Protein elution behaviour was investigated with 28 defined proteins and several protein containing preparations and biological fluids. All proteins were eluted with a neutral buffer without retardation as peaks in the permeable or excluded fraction. Due to its sharp separation limits VP can be used for the separation of proteins with small differences in size. In special cases, proteins of nearly equal molecular weight (e.g. carboxypeptidase A and pepsin) may be separated due to differences in the electrical charge of the protein molecules and resulting differences in the electric interaction with the negatively charged polygalacturonan matrix of the vesicle membrane (cell wall). Vesicle chromatography is a biocompatible process. The VP may be applied on a large scale. Complete separation between excluded and permeable proteins may be reached if the columns are loaded with concentrated protein samples (e.g., blood plasma). Size fractionation by the VP seems to be applicable in the following fields:

1. Preparative separation of an excluded protein from an excess of permeable macromolecules, especially if the difference in STOKES' diameter is too small for an effective separation by gel chromatography or conventional membrane techniques.

2. Preparative separation of permeable proteins from an excess of excluded proteins. 3. Chromatography of proteins in the presence of alcohol, polyethylene glycol or detergents.

Introduction

In the past the use of empty or polymer filled microcapsules in fixed beds for dialysis [l] or size exclusion chromatography had been proposed [2]. Up till now, however, these proposals had not received much attention; presumably because a special quality was necessary for a successful use of microcapsules as a chromatographic solid phase. The main problems to be solved for the use of microcapsules in a chromatographic mode of dialysis are the requirements of sufficient rigidity, small size and low solid content of the particles as well as defined and sharp size separation by the capsule envelope. Differences between practical results of gel permeation chromatography (GPC) and those of membrane permeation chromatography with microcapsules have only recently been demonstrated. In previous papers [3,4] it was shown that chromatographic particles representing clusters of empty and purified plant cell envelopes (vesicles) could be used ' To whom correspondence should be addressed

244 Acta Biotechnologica 12 (1992) 3

excluded macrornolecu les ds> 6.5 nrn

permeable substances ds<5.6 nm

Fig. 1. Scheme of the structure and the separation mechanism of the vesicular packing material The sue limits of exclusion and permeation are expressed as critical STOKES’ diameters 4-

for a type of exclusion/permeation chromatography. The former was designated as vesicle chromatography (VC). The stationary liquid phase within the vesicles exchanges with the mobile phase outside the vesicular particles by a diffusion process (Fig. 1). The plant cell walls are used as dialysis membranes. They are characterized by a sharp out-off limit and permit a complete separation of permeable substances from excluded macromolecules or particles. Absence of an extended size fractionation range in VC is the main difference to GPC. A detailed investigation of the sue fractionation properties of the vesicular packing material (VP) has shown that dextran molecules with a STOKES’ diameter of up to 5.6 nm equilibrate with all the stationary liquid phase and are eluted as a sharp peak with an elution volume corresponding to 95 to 97% of the bed volume. Dextran molecules with a STOKES’ diameter larger than 6.5 nm leave the column with the exclusion volume. Permeable proteins determined so far have a molecular weight lower than 30 kD. The smallest excluded protein examined [4] up till now is pepsin (M, = 35 kD) - a strongly acid phosphoprotein. The purpose of the present paper is to demonstrate the advantages of VC especially with regard to its application in the isolation of practically important proteins.

Materials and Methods

Defined Proteins and Biological Protein-Containing Fluids Tested in Vesicle Chromatography If not otherwise indicated proteins were purchased from SERVA Fine Biochemicals, Heidelberg. Trypsin zur Zellzucht (a mixture of pancreatic proteases and other hydrolases), porcine pancreatic a-amylase (contaminated with proteases) and porcine elastase were supplied from B r o c m m BERND BELGER, Kleinmachnow. Macerase from Aspergiflus niger (crude pectinolytic enzyme complex) was kindly supplied from the Forschungsinstitut fur Obst- und Gemiiseforschung, Magdeburg. Thennitase was prepared as described previously [5]. Alkaline proteinase (from Bacillus sp.) was a gift from Dr. HAFNER, Forschungszentrum fir Biotechnologie Berlin. Human blood plasma was kindly supplied by the Institut fiir Transfusiologie und Transplantologie der Charitb, Humboldt-Universitat zu Berlin.

Chromatography

The previously described vesicular packing material 131 available from the SERVA Heidelberg and PERMSELEKT GmbH Berlin was used. Chromatographic columns were prepared in the usual manner. Beds were allowed to shrink to the optimum density (about 25 mg dry packing material per ml bed volume) and then covered with filter paper or fixed between adaptors. For storage at room temperature, columns were saturated with a solution of 0.5% sodium dihydrogenphosphate (pH 5 ) containing 0.1 %

K L ~ N E , R., WOEHLECKE, H. et al., Protein Purification by Vesicle Chromatography 245

of sodium azide. The Row rate was controlled by a peristaltic pump and ranged between 0.1 and 0.2 ml per min- '. Beds packed in process columns with 25 cm I.D. were checked before usage with phenol red for satisfactory packing quality. The height equivalent of a theoretical plate (HETP) was calculated for permeable compounds from the peak variance and elution volume as described in [6].

Measurement of Enzyme Activity The unspecific proteinase activity was determined at 37 "C and pH 3.5 (pepsin) or pH 8.0 (all other proteinases) for 15 minutes using 0.4% azocasein [5]. Carboxypeptidase A was detected using 10 m M Z-glycyl-L-phenylalanine. After incubation at pH 7.5 and 37 "C the liberated phenylalanine was determined by quantitative paper chromatography. Amylase activity was determined using colorimetric measurement (578 nm) of the residual amount of starch after incubation of the sample (1 ml) for 5 min at 37 "C and pH 6.5 (0.06 M phosphate buffer). Polygalacturonidase activity was measured using 1 % pectic acid (SERVA) as substrate. After 5 min incubation of the sample (0.4 ml) at pH 4.6 and 30 "C in 0.05 M acetate buffer the reaction was stopped by the addition of 0.5 ml 0.5% 3.5-dinitro- salicylic acid (solubilized in 3.75% K/Na-tartrate and 1.2% NaOH). Subsequently the sample was heated for 5 min at 100 "C, and the resulting colour was measured at 530 nm. Lactose was measured after its hydrolysis by lactase (Novo) using a glucometer. Protein content was estimated spectrophotometrically a t 280 nm using UV-VIS-spectrophotometers (ZEISS JENA and LABORATORNI ~RISTROJE, Prague) or an UVICORD I1 photometer (LKB, Sweden).

Results and Discussion Elution Behaviour of Proteins To determine the separation limit of the VP for globular proteins, several enzymes and other native proteins with known molecular weight and isoelectric point were chromatographed on V P columns. Protein peaks were found at elution volumes of either 40 to 50% of the bed volume (excluded proteins) or 92 to 98% of the bed volume (permeable proteins). The results, indicated in Fig. 2 and Tab. 1, show that the molecular weight limit for permeation of the tested proteins lies in the range between 30 and 40 kD. Intermediate elution volumes and retardation of tested proteins caused by adsorption were not observed in 100rnM phosphate buffer at p H 7.

E,,

3

2

1

1 5.l 1.

Fig. 2. Elution diagrams of excluded (left) and permeable (right) proteins Samples: 1 - 0.5 ml of 2% ovalbumin, 2 - 0.5 ml of 2% pepsin, 3 - 1 ml of 1.5% alkaline proteinase, 4 - 1 ml of 0.9% trypsin, 5 - 1 ml of 0.5% thermolysin, 6 - 1 mI of 0.16% carboxypeptidase A. Column: 2.3 cm (i.d.) x 5 cm. Eluant: I00 mM phosphate buffer, pH 7.0 (samples 1,3 -6), pH 5.3 (sample 2). Flow rate: 0.4 ml/min. Volume of collected fractions: 1.2 ml.


Tab. 1. Behaviour of proteins in vesicle chromatography using packing material made from cell clusters of Chenopodium album*

~~ ~~~

Molecular weight Isoelectric point FDI

Excluded proteins Casein miceiles (milk, 2) 105 5.0

Aldolase (rabbit muscle, 1) 156 5.0

Hemoglobin (bovine, 1) 64.4 6.7 Malate dehydrogenase (bovine heart, 1) 63.0 6.0

Ferritin (horse spleen, 1) 450 4.1 -4.6

Albumin (human serum, 1) 67.5 4.9

a-Amylase (barley malt, 2) 56.0 5.5 a-Amylase (human saliva, 2) 53.0 5.7 a-Amylase (porcine pancreas, 2) 53.0 5.7 Albumin (hen’s egg, 1) 43.0 5.5

Pepsin (porcine, 1) 35.5 1.0 Polygalacturonase (Aspergillus, 2) 40.0 4.0

PermeabIe proteins Thermolysin (Bac. thermoproteolyticus) 35.5 8.5 Carboxypeptidase A (bovine, 1) 35.4 6.1 Subunit of MDH (bovine, 1) 31.5 6.5 Carbonic anhydrase (bovine, 1) 28.5 5.9 Thermitase (Thermoactinomyces, 2) 28.0 9.1

Elastase I (porcine, 1, 2) 25.7 9.5 Chymotrypsinogen (bovine, I ) 25.6 9.5 Trypsin (bovine, 1) 23.3 10.2 Myoglobin (horse, 1) 17.8 7.3 Lysozyme (egg white, 1) 14.3 11.0

Ribonuclease (bovine, 1) 13.7 9.3

Alkaline protease (Bac. sp., 2) 28.0 9.1

a-Lactalbumin (bovine, 2) 14.2 5.6

Cytochrome C (bovine, 1) 12.2 10.6

* A11 compounds were chromatographed in 100 mM phosphate buffer, pH 7.0-pH 7.3 (with the exception of casein micelles: acetate buffer pH 4.6). (1): SERVA, Heidelberg, (2): crude preparations.

Separation of Pepsine from Carboxypeptidase A

Fig. 3 shows the complete separation of two nearly equal-sized proteases, pepsin (35.5 kD) and carboxypeptidase A (34.5 kD). This result suggests that not only the size but also the electrical charge of protein molecules determines their permeability. Pepsin (IEP = 1.0) has a much higher negative charge than carboxypeptidase A (IEP = 6.1). The cell wall is a negatively charged polysaccharide membrane, the separation properties of which are controlled by the polygalacturonan matrix. With regard to the diffusion of a poiyanion, the effective hydrodynamic radius of the macromolecule, which is essential for total or partial exclusion from a negatively charged matrix, is composed of the STOKES’ radius (rs) and an electrical barrier of thickness d [I. The dependence of d on the electrical charge might explain the finding that pepsin (rs = 2.3 nm) is excluded whereas dextran molecules with rs = up to 2.8 nm [4] as well as less negatively or positively charged protein molecules with a size similar to pepsin penetrate the vesicle membranes (Tab. 1).

KLEINE, R., WOEHLECKE, H. et al., Protein Purification by Vesicle Chromatography 247

2' Fr. Na I 0

Fig. 3. Separation of pepsin from carboxypeptidase A with vesicle chromatography Activity of proteinases in eluate fractions was detected specifically as described in materials and methods. Sample: 5 mg of pepsin (porcine) and 1.8 mg of carboxypeptidase A (bovine) in 1 ml of the eluant. Column: 2.3 cm (i.d.) x 5 cm. The mixture of both enzymes was chromatographed at two different pH values: pH 7 for subsequent determination of the carboxypeptidase activity (curve l), pH 6 for subsequent determination of the pepsin (curve 2). Flow rate: 0.4 ml/min. Volume of collected fractions: 1.2 ml.

Generally, size fractionation of proteins by the V P can only roughly be characterized by a critical molar mass of exclusion; as the shape, charge and carbohydrate content of protein molecules might be responsible for their hydrodynamic behaviour in the vesicle membrane.

Separation of Proteins in Detergent-Containing Media

Among the various detergents used in GPC of proteins sodium dodecyl sulfate (SDS) at a concentration of more than 0.05% w/v proved to cause significant shrinkage of Sephadex gels with concomitant alteration of elution volumes [S, 91. In contrast, for the VP no measurable change of bed and elution volumes could be observed for the VP in the presence of 0.1% SDS. Fig. 4 shows the elution peaks of dimeric (excluded) and monomeric (permeable) malate dehydrogenase. Rechromatography demonstrates the potency of VC for the isolation of subunits of oligomeric proteins. Similar results (not shown) were obtained with either 1% sodium taurocholate or 6 M urea.

Chromatography of Ethanol Soluble Proteins

The V P may also be used in buffers containing ethanoI in high concentrations. Due to the cellular structure of the packing particles, the bed volume is not reduced by dehydration. Therefore, the VP may be an interesting tool for size fractionation of membrane proteins

Acta Biotechnologica 12 (1992) 3

0.6 “1 1.5

Fig. 4. Vesicle chromatography of bovine heart rnalate dehydrogenase (MDH) in the presence and absence of sodium dodecyl sulfate (SDS) Right part: Fractionation of native and denaturated samples. Native sample: 10 mg of MDH in 1 ml eluant (full line). Denaturated sample: 10 mg of MDH in 1 rnl eluant containing 1% SDS and 1% p-mercaptoethanol (dotted line). Column: 2.3 cm (i.d.) x 5 an. Eluants: 100 mM phosphate buffer, pH 7.2 (full line) and 100 mM phosphate buffer, pH 7.2 containing 0.1% SDS (dotted line). Flow rate: 0.4 ml/rnin. Detection: 1.2 ml of the fractions were collected and registered spectrophotometrically at 278 nm. Left part: To demonstrate the purity of the separated fractions, peak fractions of the separation shown on the left (fraction number 6 of native MDH and number 15 of denaturated MDH) were rechromatographed separately under identical conditions.

Fig. 5. Chromatography of ethanol soluble proteins from Zea mays seeds Sample: 1 rnl of a zein fraction obtained by extraction of defatted maize flour with 70% ethanol. Column: 2.8 crn (i.d.) x 8.6 cm. Eluant: 70% ethanol. Flow rate: 0.6 ml/min. Detection: optical density at 280 nm.

KLEINE, R., W O ~ C K E , H. et al., Protein Purification by Vesicle Chromatography 249

and other ethanol soluble proteins. Using 70% ethanol as solvent, the maize prolamines (zeins) with molecular weights between 14 and 27 kD [lo] are eluted in the permeable fraction (Fig. 5).

Isolation of a-Amylme from a Mixture of Pancreatic Hydrolases

A mixture of soluble enzymes of porcine pancreas (“trypsin” preparation used for animal tissue culture) may be divided on a 9 cm column of the VP into an excluded fraction (about 4% of the whole preparation) and a large permeable fraction (Fig. 6)- The latter consists of a mixture of proteases (trypsin, chymotrypsin, elastase, carboxypeptidases) and salts, whereas the excluded fraction contains the highly purified and active a-amylase (both isoenzymes) free from protease (electrophoretic data and enzyme activities not shown). Conversely, it is also possible to obtain the protease group (especially elastase and trypsin) almost free from a-amylase. For comparison, the pancrease hydrolase preparation was fractionated on a 20 cm column of Sephadex G 50 fine. In spite of the larger bed height of the GPC column the amylase peak was not clearly separated from the main protein fraction (chromatogram not shown). The advantage of VC in comparison with GPC in these and the following applications is due to the large difference between relative elution volumes of permeable and excluded proteins.

i 30 sb ml

Fig. 6. Separation of a-amylase (small peak) from pancreatic proteinases and other solutes (large peak) Sample: 125 mg of the pancreatic preparation “Trypsin zur Zellzucht” in 2.5 rnl of the eluant. Column: 2.9 cm (i.d.) x 9 cm. Eluant: 10 m M phosphate buffer, pH 7.0, 100 rnM NaCI. How rate: 1 ml/min. Ordinate: refractometric index (arbitrary units).

Fractionation of the Macerase-Enzyme Complex

Although the VP consists of primary plant cell walls which may be completely dissolved by pectinolytic enzymes, this material can be applied to purification of polygalacturonase if the enzyme activity is reversibly inhibited. Fig. 7 shows the elution diagram of a crude


Fi Q

PG w 60

40

20

10 20 Fr. Na

Fig. 7. Fractionation of “Macerase” (pectinolytic enzyme complex) Sample: 0.25 ml of the crude enzyme preparation. Column: 2.3 cm (i.d.) x 6.5 cm. Eluant: 50 mM Tris/HCI, pH 7.3 (free from calcium). Flow rate: 1 ml/min. Collected fractions: 1.2 ml. Detection: solute concentration (curve 1) refractometrically, polygalacturonase (PG) activity (curve 2), see Materiat and Merhods, UV absorbing material (curve 3) by spectrophotometer a t 280 nm.

pectinolytic preparation at pH 7.3 in the absence of Ca2+ ions. All the polygalacturonase activity was eluted with the exclusion volume whereas the lower molecular weight contaminants (proteins, peptides, sugars, nucleotides etc.) appeared in the larger second peak fraction.

Fractionation of Milk Components

The separation of the casein-milk fat micelles from lactose and whey proteins is shown in Fig. 8. It can be seen that the casein fraction of milk was separated quantitatively from lactose and lactalbumine. All other whey proteins were eluted with the excluded fraction. On the other hand, this method allowed the preparation of casein-lipid-free lactose solutions.

Separation of a Low Molecular Weight Protein Fraction from Undiluted Human Blood Plasma

Beds of the VP may be loaded with relative large volumes of biological fluids containing proteins in high concentration. The separation of a small fraction of permeable proteins from undiluted blood plasma (Fig. 9) demonstrates the high loading capacity of columns used for VC. The permeable fraction (minor peak) contained several proteins (precipitable by ammonium sulfate) with molecular weights below 40 kD (proved by disc electrophoresis). The fractionation shown in Fig. 9 was repeated several times with the same column without any significant changes in the elution diagram. Obviously, the vesicle membranes were not altered by highly concentrated proteins. VC surpasses other membrane separation techniques owing to its large specific membrane surface and extremely high membrane permeability. As the membrane transport not volume flow but diffusion, membrane fouling seem to be no problem in VC.

KLEINE, R., WOEHLECKE, H. eta!., Protein Purification by Vesicle Chromatography 251

10 20 Fr. NQ

Fig. 8. Fractionation of milk Sample: 1 ml of fresh cow’s milk. Column: 3.2cm (i.d.)x 1Ocm. Eluant: 100 mM Na-acetate buffer, pH 6.1, containing 6.5 mM CaCl,. Flow rate: 0.5 ml/min. Collected fractions: 5 ml. Detection: solute concentration (curve 1) by differential refractometer RIDK, lactose (curve 2) see Material and Methods, UV absorbing material (curve 3) by spectrophotometer at 280 nm.

Fig. 9. Fractionation of human blood plasma proteins Sample: 2.5 ml of human citrate plasma. Column: 2.52 cm (i.d.) x 10 cm. Eluant: 10 mM phosphate buffer with 50 mM NaCI, pH 7.0. Flow rate: 0.7 ml/min. Detector: spectrophotometer LCD 2040 (280 nm).

Suitability of the V P for Application in a Process Scale

The scaling up of VC was studied in a commercial process column. An optimum packing density (25 to 28 g dry material per 1 bed volume) was found to be important, especially for the use of large preparative columns. Even with a large sample and bed volume a bed


height of 9.5 cm was found to be suficient for complete separation of excluded proteins from permeable ammonium sulphate or polyethylene glycol (Fig. 10). Chromatographic efficiency, determined as height equivalent of one theoretical plate (HETP) of the permeable fraction was practically the same as in columns of a smaller diameter. In the separations shown in Fig. 10, HETP for ammonium sulphate was 0.21 mrn and for polyethylene glycol was (4000 kD) 0.4 mm. The demonstration of complete separation of polyethylene glycol (PEG) from excluded protein molecules is a relevant example of application of the VP with respect to the great importance of PEG as a means for protein purification by liquidniquid extraction [lo] and PEG precipitation [ll]. The scaling up of VC should generally be carried out by increasing the bed diameter and not the bed height. Prolongation of the separation distance above the value necessary for a base line separation will increase dilution of separated fractions and separation time. Use of extended beds at high flow rates is unfavourable as the VP is susceptible to compression by the existing pressure gradient. The optimum flow rate is also limited by the time necessary for diffusion of permeable compounds within the particles. In order to

HSA PEG 4000

2 4 6 Iitres

2 4 litres 6

Fig. 10. Large scale separations Samples: upper part - 2 g of polyethylene glycol 4000 (PEG) and 1 g of human serum albumin (HSA) in 200ml of eluant; lower part - 2 g (NH4)2S04 and 1 g ovalbumin (egg) in 200 ml of eluant. Column: Pharmacia BP 252, 25.2 cm (i.d.) x 9.5 cm. Eluant: 100 mM phosphate buffer, pH 7.0. Flow rate: 40 mI/min. Detector: differentia1 refractometer RIDK 101.

KLEINE, R., WOEHLECKE, H. et al., Protein Purification by Vesicle Chromatography 253

prevent kinetically caused peak broadening in the separation of permeable macromolecules the flow rate should not be much higher than 10111Icm-~ h-' from excluded ones (unpublished results). The application field of VC will be extended by the development of VP with different separation limits. The materials produced by altering the porosity of the vesicle membranes (cell walls) will be described in a following paper.

Conclusion

Chromatography with the cell structured or vesicular packing material, respectively, seems to be an interesting alternative to gel fdtration or conventional membrane separation for preparative downstream processing of biological fluids. One specific advantage is the large distance of separated peaks and the sharp separation limit. Size fractionation by VC seems to be suitable for the following fields of application:

1. Separation of excluded proteins from an excess of permeable molecuies, especially if the difference in the STOKES' diameter is too small for an effective separation by GPC or conventional membrane techniques.

2. Separation of permeable proteins from an excess of excluded ones. 3. Purification of proteins in samples containing alcohol, polyethylene glycol or detergents

and the use of solutions of these compounds as eluant materials.

Received June 12, 1991

References

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[4] EHWALD, R., HEESE, P., KLEIN, U.: J. Chromatogr., in press. [5] KLEINE, R.: Acta Biol. Med. Germ. 42 (1982), 89. [6] GL~CKNER, G. : Polymercharakterisierung durch Fliissigchromatographie. Berlin: VEB Deutscher

[7] DUBIN, P. L., SPECK, C. M., KAPLAN, J. I.: Anal. Chem. 60 (1980), 895. [8] FISH, W. W. J.: Agric. Food Chem. 19 (I971), 660. [9] FISH, W. W., MA", K. G., TANFORD, C.: J. Biol. Chem. 244 (1971), 4989.

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[lo] WALLACE, J. C., LOPES, M. A., PAIVA, E., LARKINS, B. A.: Plant Physiol. 92 (1990), 191. [l I] KULA, M.-R. - In: Comprehensive Biotechnology. Ed.: MOO-YOUNG. Oxford, New York,

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application of vesicle chromatography in protein purification

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