affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

Affinity Precipitation and Site-Specific Immobilization of Proteins Carrying Polyhistidine Tails

Jonas Carlsson, Klaus Mosbach, and Leif BUlow* Department of Pure and Applied Biochemistry, Chemical Center, POB 124, S-22 1 00 Lund, Sweden; e-mail: Leif [email protected]

Received July 8, 1995/Accepted February 8, 1996

Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), 0-glucoronidase (Escherichia cold, and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in f. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycol- bis (P-aminoethyl) tetraacetic acid (EGTA) charged with Zn2+ as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn2+. The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity. 0 1996 John Wiley & Sons, Inc. Key words: metal affinity precipitation polyhistidine tails protein immobilization

INTRODUCTION

Today, there are a number of chromatographic methods available for purification of recombinant proteins carrying genetically introduced affinity tails (for reviews, see refs. 16 and 21). A widely used method for protein purification embraces affinity tails with metal binding properties and immobilized metal affinity chromatography (IMAC).'3,20,23 A number of different amino acid residues can contribute to metal binding, but particularly histidine residues are known to form strong complexes with Zn2+, Cu2+, Ni2+, and Co2+. Affinity tails composed of ( h i ~ ) ~ - ~ , 5 - ~ (his-trp)l,3,18 and (his-gly-his)," have thus all been explored. Although chromatographic procedures are excellent for isolating proteins on an analytical scale, they are, in many instances, unsuitable for large scale purifications.

In contrast, protein precipitation offers not only a more simple and rapid procedure, but it is also most

* To whom all correspondence should be addressed.

often less expensive. A major drawback of protein precipitation is the low degree of purity frequently obtained in a single step. By affinity precipitation with bifunc- tional ligands, introduced by Larsson and Mosbach in 1979,8 it is often possible to improve the purity of the final product. However, for many proteins no suitable affinity ligand exists and, if it does, it is frequently difficult to synthesize chemically. An attractive solution is affinity precipitation of genetically modified proteins with, e.g., metal binding properties. Lilius et al.9 have previously reported on the purification of a recombinant galactose dehydrogenase carrying five histidine residues in the amino-terminus of the protein, galDH(his)5, by precipitation using ethylene glycol-bis(p-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) charged with Zn2+.

In this study, we have improved and further evaluated the potential of affinity precipitation of various proteins carrying polyhistidine tails with EGTA(Me)2. EGTA can coordinate two metal ions per molecule. The technique requires at least two metal binding sites on the protein for aggregation to occur. This implies that oligomeric proteins with one attached affinity tail on each subunit may be purified using this method. The precipitation of a tetrameric protein carrying four histidine residues, one on each subunit, is schematically illustrated in Figure 1. On the other hand, monomeric proteins carrying only a single polyhistidine tail should not be able to form large protein aggregates, i.e., no precipitation would occur. The proteins examined in this report are lactate dehydrogenase (Bacillus stearothermophifus) (LDH), P-glucoronidase (Escherichia cofi) (p-glu), galactose dehydrogenase (Pseudomonas Puorescens) (galDH), and protein A (Staphylococcus aureus). Poly- histidine tails were genetically inserted into these proteins either in the amino- or carboxy-terminus. Zn2+ or Cu2+ were chelated to EGTA and the influence of various experimental conditions for precipitation was investigated. For comparison, the behavior of these proteins on IMAC using iminodiacetic acid (IDA) as chelating ligand was examined.

We were also interested in determining the potential of using the same polyhistidine tails for protein immobi-

Biotechnology and Bioengineering, Vol. 51, Pp. 221-228 (1996) 0 1996 John Wiley & Sons, Inc. CCC 0006-3592/96/02022 1-08

Figure 1. Schematic illustration of metal affinity precipitation of a tetrameric enzyme carrying four genetically attached polyhistidine tails.

lization. Immobilized enzymes are reusable and frequently more stable than their free counterparts.’* Un- fortunately, many immobilization methods are quite unspecific and result in significant losses of enzyme activity.” Furthermore, many enzymes lack suitable amino acid residues for chemical coupling procedures. Genetically introduced polyhistidine tails may therefore be a valuable complement for site-specific and reversible immobilization of proteins on IMAC gels.

MATERIAL AND METHODS

Chemicals and Reagents

Restriction enzymes, T4-DNA ligase, NAD, NADH and isopropyl-P-D-thiogalactopyranoside (IPTG) were supplied by Boehringer Mannheim. p-Nitrophenyl glu- coronide (PNPG), EGTA, bovine serum albumin (BSA), 2,2’-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and molecular weight standard proteins for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were from Sigma. Protein A and o-phenylenediamine (OPD) were obtained from Pierce. Rabbit anit-mouse immunoglobulins linked to horseradish peroxidase and microtiter plates were from Da- kopatts and Nunc, respectively. Chelating Sepharose 6B was purchased from Pharmacia. Acrylamide and kits for protein quantifications and silver staining were from BioRad.

Bacterial Strain and Plasmids

E. coli strain F’11 recA ((lac, pro) Athi, rifA, strA, recA/F’lacIqZ-, pro+)14 was used as bacterial host in

all experiments. The plasmids (pDH101; pBT42H8: pUL1841: and ~ U H L 1 8 4 2 ~ expressing the structural genes for galDH (Pseudomonas JEuorescens), galDH- hi^)^, LDH (Bacillus stearothermophilus) and LDH- hi^)^, respectively, have been described previously. The structural gene of p-glu (Escherichia coli) was obtained from ~ R A J 2 7 5 G p l . ~ Plasmid pRIT2T expressing a modified intracellular protein A (Staphylococcus aureus) was purchased from Pharmacia.

Plasmid Construction

To prepare plasmids expressing /3-glu( his)4 and protein A ( h i ~ ) ~ four oligonucleotides were synthesized at the molecular biology core facility (Lund University). The oligonucleotides 5’-TCG ACC ATG CAT CAC CAT CAC GC-3’ and 5’-CAT GGC GTG ATG GTG ATG CAT GG-3’, which form a linker encoding four histidine residues, were inserted in the 5’-end of the p-glu gene. A polyhistidine tail, composed of the oligonucleotides 5’-AAT TAC GTG CAC CAT CAC CAT CAC TAG-3’ and 5’-TCG ACT AGT GAT GGT GAT GGT GCA CGT-3’, encoding five histidine residues was inserted in the 3‘-end of the protein A gene. The oligonucleotides were mixed and heated to 70°C for 10 min and then slowly cooled to room temperature to allow hybridization. All other cloning procedures were performed as described by Sambrook et al.l5

Cell Culture and Harvesting

Cultures grown to late exponential phase in LB broth15 supplemented with 50 mg/L ampicillin and 0.1 mM IPTG (when needed), were harvested by centrifugation, washed, and resuspended in 50 mM sodium phosphate buffer (pH 7.5) containing 0.1 M NaCl and 1 mM dithiothreitol (DTT) (buffer A). After sonication and centrifugation, solid ammonium sulfate was added to the supernatant. The fraction between 35% and 65% satura- tion was collected and dissolved in buffer A.

Enzyme Assays

Enzyme activities were recorded spectrophotometri- cally at 20°C. The LDH activities were monitored at 340 nm in 50 mM sodium phosphate buffer (pH 6.3) containing 0.2 mM NADH and 30 mM p y r ~ v a t e . ~ The galDH activity measurements were performed in 0.1 M Tris-HC1 (pH 8.5) containing 0.1 M NaCl, 16 mM galactose, and 0.2 mM NAD.’ The p-glu activities were measured at 415 nm in 50 mM sodium phosphate (pH 7.0) supplemented with 1 mM EDTA and 1 mM PNPG.3

Protein A Analysis

Protein A was quantified using a simple enzyme-linked immunosorbent assay (ELISA). Samples and standards

222 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996

were diluted in 0.1 M sodium phosphate buffer (pH 9.5) and were allowed to coat microtiter wells for 2 h at 37°C. Remaining binding sites were blocked with 0.1 mg/mL BSA in 25 mM sodium phosphate buffer (pH 8.0), 0.5 M NaC1, and 0.1% Tween-20 for 20 min at room temperature. The wells were then incubated for 1 h at 37°C with horseradish peroxidase labeled antibodies. The microtiter plates were washed four times with 0.9 M NaC1,0.05% Tween-20 between each step. Finally, the substrate (1 mM ABTS and 0.1 pL/ mL 39% hydrogen peroxide in 50 mM sodium citrate pH 4.0) was added to the wells and the absorbance read at 405 nm.

Protein Determinations and Electrophoresis

Protein concentrations were determined by the BioRad protein microassay procedure using BSA as standard. SDS-PAGE was performed on 15% polyacrylamide slab gels using a Tris-glycine (pH 8.3) discontinuous buffer system according to Laemmli?

Metal Affinity Precipitation

The metal chelate complex EGTA(Me)2, was prepared by mixing 2.5 mmol EGTA and 5.2 mmol K2C03 in 50 mL of distilled water.” EGTA was assumed to coordinate two metal ions but added in excess to avoid inhibition of unchelated metal ions. Thus, by addition of 4.8 mmol CuC12 or ZnC12, 96% of the binding sites were occupied. Protein samples to be precipitated were dialyzed and diluted to a protein concentration of 1 mg/mL in the desired precipitation buffer. Two vol- umes of diluted protein and one volume of EGTA- (Me)z complex were mixed by adding the chelating agent drop by drop. The solution was incubated on ice for 20 min, pelleted by centrifugation, and washed twice with 50 mM ice-cold sodium acetate buffer pH 6.0. Finally, the pellet was dissolved in 0.1 M Tris-HC1 buffer (pH 7.5) containing 30 mM EDTA or in 50 mM sodium phosphate buffer (pH 6.2) containing 0.3 M imidazole. Undissolved precipitate was removed by centrifugation prior to protein and enzyme activity determinations.

Protein Purification Using IMAC

The IMAC gel was loaded with Zn ions according to the instructions of the manufacturer. The column (0.7 X 12 cm) was equilibrated with 50 mM sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl and 1 mM DTT (buffer B). One milliliter of protein solution was then applied. Bound protein was eluted with a 60 mL pH gradient ranging from 7.5 to 4.5 using 0.1 M Tris-acetate buffer supplemented with 0.5 M NaCl and 1 mM DTT (buffer C). Elution was also performed at pH 7.5 using an imidazole gradient ranging from 5 to 300 mM in

buffer C. All steps were carried out at 4°C using a flow rate of 0.4 mL/min.

Studies of Site-Specific Immobilization on Chelating Sepharose

All immobilization experiments were carried out in a batch mode at 4°C. One milliliter of Zn2+- or Cu2+- loaded gel suspension, corresponding to 0.2 mL of moist gel, was pipetted into a 10-mL polystyrene test tube. The gel was equilibrated with buffer B and mixed with the crude protein solution obtained after ammonium sulfate precipitation. The tube was gently rotated for 15 min. Finally, the gel was washed with 3 X 10 mL buffer B. Before discarding the supernatant, the tube was centrifuged for 20 s. Prior to enzyme activity measurements the gel loaded with proteins was slowly heated to room temperature and the buffer changed for enzyme monitoring. P-Glu activities were registered in 50 mM Tris-HC1 (pH 7.5) supplemented with 1 mM PNPG, LDH activities were determined in 50 mM sodium phosphate buffer (pH 6.7) supplemented with 0.2 mM NADH and 30 mM pyruvate; galDH was determined in 50 mM sodium phosphate buffer (pH 7.5) containing 0.2 mM NAD; and finally, horseradish peroxidase was monitored in 50 mM sodium phosphate buffer (pH 6.6), 0.4 mg/mL OPD, and 0.4 pL/mL hydrogen peroxide. The leakage of immobilized protein was estimated by several changes of substrate buffer.

RESULTS

Vector Construction and Gene Expression

Figure 2A illustrates the introduction of DNA fragments encoding polyhistidine tails to the 3‘-end of the protein A gene and the 5’-end of the p-glu gene, respectively. The plasmid pRIT2T carrying the protein A gene was digested with EcoRI and PstI, and a DNA linker encoding five histidine residues was inserted. Similarly, a linker encoding four histidines was introduced into the P-glu plasmid pRAJ275GP1 previously cut with SalI and NcoI. The vectors were expressed in E. coli F’11 and were routinely grown to late exponential phase. The DNA and amino acid sequences surrounding the histidine tails of these and the galDH and LDH plasmids used in this study are depicted in Figure 2B.

Metal Affinity Precipitation

To optimize the experimental conditions for protein precipitation with EGTA(Zn)2, the infl!ience of pH and ionic strength was initially investigated using L D H ( ~ ~ s ) ~ as a model protein. L D H ( ~ ~ s ) ~ is expressed at high levels in E. coli and can easily be absorbed on IMAC columns charged with Zn2+ Furthermore, it is a thermostable

CARLSSON, MOSBACH, AND BULOW: METAL AFFINITY PROTEIN PRECIPITATION 223

\ [ / G TCG ACC ATG GGC CCG

pRIT2T

8GGCCCGGCCGGGCCC

B Protein A' R QGlu I AmpR I AmpR

EcoRl ApaLl Psfl Sall Nsll Ncol

Inserted linker Inserted linker

GGG CCC ATG GTC

Pro Gly Asn Tyr Val His His His His His Stop 5' - Protein A - CCG GGG AAT TAC GTG CAC CAT CAC CAT CAC TAG - 3

M e / His His His His Ala Me/ 5' - ATG CAT CAC CAT CAC GCC ATG - m- 3

Tyr Asp Gly Asp His His Hjs His His Stop 5' - GalDH - TAC GAT GGG GAT CAT CAC CAT CAC CAT TAA - 3'

M e t Thr M e t Ile Thr Asn Ser His His His His- 5' - ATG ACC ATG ATT ACG AAT TCA CAC CAT CAC CAT-

Gly Asp Pro Ala Met GGG GAT CCG GCA ATG - !J!j - 3

(b) Figure 2. (a) Construction of plasmids. Plasmid pRIT2T encoding protein A' was digested with EcoRI and PsN. Similarly, pRAJ275GP1 carrying the p-glu gene was cleaved with Sun and NcoI. Two different DNA linkers encoding polyhistidine tails were inserted into the plasmids. (b) DNA and amino acid sequences of the histidine linkers. The DNA sequence and the corresponding amino acid sequences surrounding the polyhistidine tails of protein A(his)s, P-glu(his)4, galDH(hi~)~, and LDH(his)4 are illustrated. The histidine linkers are inserted in the amino-terminal ends of P-glu(hi~)~ and LDH(his)4, and in the carboxy-terminus of protein A ( h i ~ ) ~ and gal- D H ( ~ ~ s ) ~ .

enzyme and its activity can be monitored without diffi- culty. The pK, values for surface histidines are normally between 6 and 7. As evident from Table I (part A), the optimal pH for precipitation was also found to be 7.0 indicating that the histidine residues most probably are located on the surface. The use of high ionic strength is known to improve separations on IMAC.22 As indicated in Table IB the degree of purification obtained after precipitation is improved utilizing buffers with in- creasing NaCl concentrations. However, the yield of purified protein is reduced at high ionic strength.

Metal affinity precipitation of native and engineered LDH, galDH, p-glu, and protein A at different concentrations of EGTA(Zn)2 are presented in Figure 3. The precipitation procedure was followed by determining the

Table I. Influence of pH (A) and NaCl (B) on metal affinity precipitation of L D H ( ~ ~ s ) ~ . =

NaCl Final yield Purification PH ( M I pellet (%) factor

A 6.0 6.5 7.0 7.5

B 7.0

0.5

0 0.1 0.3 0.5 1.0

22 43 68 58 59 71 71 68 56

18 20 22 18 16 19 20 22 23

aLDH(his)r was precipitated in 50 mM sodium phosphate buffer and the pellet was dissolved in 50 mM sodium phosphate (pH 6.2) containing 0.3 M imidazole.

enzyme activity both in the supernatant and in the dissolved pellet. Obviously, all proteins aggregated better if they were carrying a polyhistidine tail. At a final concentration of 3.3 mMEGTA(Zn)2, enzyme recoveries corresponding to 70% for L D H ( ~ ~ s ) ~ and 68% for galDH(hi~)~ were achieved. By a single affinity precipitation step, it was possible to purify L D H ( ~ ~ s ) ~ and galDH(hi~)~ by a factor of 22 and 25, respectively (Table 11).

Without exception, addition of more chelating agent resulted in higher yields but lower purity because the native protein also started to precipitate. For instance, when LDH(his)4 was precipitated in 8.3 mM EGTA(Zn)*, the recovery was 82% but the enzyme was purified only six-fold. The protein recoveries were followed using activity balances which indicated that less than 10% of the protein precipitate failed to redissolve. However, recoveries could be improved if the final washing step of the precipitate was omitted, but the purification values given in Table I1 were then routinely decreased by a factor 1.5 to 2. In general, the loss of enzyme activity in the washing steps was estimated to be below 20%. L D H ( ~ ~ s ) ~ was also precipitated directly from a crude protein extract. As indicated in Table 11, the protein could be purified 24-fold. For comparison, L D H ( ~ ~ s ) ~ was also purified by affinity chromatography using oxamate Sepharose." When these two protein samples were examined by SDS-PAGE it was obvious that a simple precipitation step could give approxi- mately the same degree of purification as a conventional chromatographic procedure.

The recovery rates of protein A ( h i ~ ) ~ and p-glu(hi~)~ were not as a successful as for galDH(hi~)~ and LDH- hi^)^. In contrast to other enzymes, protein A is a mono- mer with a single histidine tail which influences its likeli- hood to form protein complexes necessary for affinity precipitation. On the other hand, p-glu is strongly inhib- ited by metal ions such as zinc and copper. At 3 mM EGTA(Zn)2, no activity of p-glu or p -g l~ (h i s )~ could be detected in the supernatant although the yield in the


100 t I

0.2 0.5 1.3 3.3 8.3 Final concentration EGTA(Zn) (mM)

loo 7

0.2 0.5 1.3 3.3 8.3

Final concentration EGTA(Zn) (mM)

loo I

Q :.:. . .:. .: . .:;.

$ 4 1 , ~, m,J,J .:;. ..::. . .:. :::

: ., . ,' :; . . '(. : .:. . ., . .;:.

. . . . 20

:.: : :. .I'. ... 0.2 0.5 1.3 3.3 8.3

Final concentration EGTA(Zn) (mM)

100 7

Protein A Protein A(his)5

P

. ; '. '"6 . , , -,J-

0.2 0.5 1.3 3.3 8.3

Final concentration EGTA(Zn), (mM)

Figure 3. Metal affinity precipitation of galDH, LDH, B-glu, and protein A, native and polyhistidine-modified proteins, respectively. Ammonium sulfate fractions were dialyzed and diluted to 1 mg X mL-' total protein in 50 mM sodium phosphate (pH 7.0) containing 0.5 M sodium chloride and 1 mM dithiothreitol. Samples were precipitated at various final concentrations of EGTA(Zn)2. The pellets of galDH, galDH(hi~)~, protein A, and protein A(his)5 were dissolved in 0.1 M Tris-Hcl (pH 7.5) containing 30 mM EDTA, while the pellets of LDH, L D H ( ~ ~ s ) ~ , p-glu, and p-glu(his)4 were dissolved in 50 mM sodium phosphate buffer (pH 6.2) supplemented with 0.3 M imidazole.

pellet was low. This latter behavior was specific for the p-glu enzymes and was probably a result of metal ion inhibition.

Purification Using Chelating Sepharose 6B

To determine the metal binding properties of the proteins used in the precipitation experiments, as well as

Table 11. Enzyme recovery after ammonium sulfate and metal affinity precipitation using 3.3 mM EGTA(Zn)2.

Number of Final yield Purification Enzyme protein subunits in pellet (%) factor

L D H ( ~ ~ s ) ~ 4 68a 22 L D H ( ~ ~ s ) ~ 4 64" 24 p-Glu( 4 35" 14 GalDH(his)5 2 70' 25

- Protein A ( h i ~ ) ~ 1 2'

"Protein pellet dissolved in 50 mM sodium phosphate containing

bMetal affinity precipitation of a crude cell extract. 'Protein pellet dissolved in 0.1 M Tris-HC1 (pH 7.5) containing

0.3 M imidazole.

30 mM EDTA.

to verify the integrity of the fusions, the native and the engineered enzymes were applied to a column of chelating Sepharose 6B. Native galDh and LDH did not bind to a zinc-loaded IMAC column. In contrast, galDH(his)5 and LDH(his)4 were completely absorbed and could be eluted at pH 5.8 and 5.5, respectively. Protein A did not bind to chelating Sepharose loaded with Zn2+ but could partly be purified if zinc was replaced by copper (also, cf. ref. 10). In contrast, protein A(his)5 could easily be adsorbed on a Zn+-charged IMAC gel.

When using a linear pH gradient, protein A(his)5 was eluted at pH 5.3 with a 95% recovery. Because p-glu was able to bind metal ions, both the native and histidine- modified enzyme were found to interact with an IMAC gel loaded with Zn2+. The pH of elution was 6.0 and 5.8 for p-glu and /3-glu(his)4, respectively. Unfortu- nately, both enzymes lost 90% of their activity after elution probably due to zinc ions bleeding from the gel during the pH gradient. The addition of EDTA to the eluted fractions did not affect the recovery. However, at least 90% of the activity was preserved when the enzymes were eluted with imidazole. Using the latter

CARLSSON, MOSBACH, AND BULOW: METAL AFFINITY PROTEIN PRECIPITATION 225

method, p-glu was eluted at 40 mM and p-gl~(his)~ at 60 mM imidazole. When examined by SDS-PAGE, p- glu was found to be less pure than P-glu(hi~)~ (data not shown). The purification factor was 15 for the native enzyme and 110 for the modified histidine.

Immobilized Proteins

We were also interested in determining the enzymatic activities of LDH(his)4, galDH(hi~)~, P-gl~(his)~, and the complex between protein A ( h i ~ ) ~ and horseradish peroxidase labeled immunoglobulins when the proteins were absorbed on chelating Sepharose 6B charged with either Zn2+ or Cu2+. The relative (immobilized vs. soluble) activities for the enzymes are presented in Table 111. To facilitate the monitoring of activities these experiments were performed in a batched mode. All enzymes were active when immobilized. The immobilized conju- gate protein A(his)5-IgG enzyme displayed the same activity as soluble peroxidase labeled IgG using Cu2+ as ligand (also, cf. ref. 10). Due to protein bleeding from the matrix LDH(his)4 could only be bound to Cu2+ charged chelating Sepharose and then exhibited a relative activity of 83%. Furthermore, the relative activities of the tetrameric enzymes L D H ( ~ ~ s ) ~ and P-gl~(his)~ were higher than that of the dimeric galDH(hi~)~. In the cases of galDh(hi~)~ and P-gl~(his)~, the enzymatic activities were higher if zinc instead of copper was used as chelating metal.

The tendency of the proteins to bleed from the gel was also investigated by measuring remaining enzyme activity on the gel after several changes of buffer (Table 111). The activities of galDH(hi~)~, L D H ( ~ ~ s ) ~ , and p- his)4 were almost completely preserved when Cu2+ was used as metal ligand. However, only galDH(hi~)~ and unconjugated protein A ( h i ~ ) ~ were stable when Zn2+ replaced the copper ions. The long-term stability of the immobilized enzymes were also investigated by storing the tubes containing soluble and immobilized enzymes

Table 111. Catalytic activity of polyhistidine-modified enzymes adsorbed on chelating Sepharose 6B.

Recovered Metal activity" Retained

Enzyme ligand (%) activityb ~

GalDH( his)g c u

LDH( c u

P-Glu( his)4 c u

Protein A(his)5-Ig-HRP c u

Zn

Zn

Zn

Zn

11 89 46 91 83 90 NB - 33 77' 40* 95 85 96' 2oE

-

NB = no binding to the resin in batch procedure. "Catalytic activity of adsorbed versus soluble enzymes. bRetained activity of adsorbed enzymes after 20 buffer changes. CSubstantial enzyme bleeding from the resin.

Table IV. Stability of immobilized versus soluble enzymes.

Stability Stability Metal immobilized" solubleb

Enzyme ligand (%) @I 32 GalDH -

GalDH(his)5 Zn 24 24 55 LDH -

P-Gh Zn 70 82 ,f3-Glu( Zn 88 51

-

- L D H ( ~ ~ s ) ~ c u 67 45

"Retained enzyme activity after storage for 12 days at +4"C on

bRetained enzyme activity after storage for 12 days at +4"C in chelating Sepharose 6B.

ammonium sulfate suspension.

at 4°C for 12 days (Table IV). All of the immobilized enzymes displayed activities in the same range or higher than the soluble enzyme. In these experiments, D'IT was not used because it promoted bleeding of the proteins from the gel.

DISCUSSION

Affinity tails have become an attractive tool in biotechnology to facilitate downstream processing of proteins after fermentation. The aim of this work has been to evaluate the usefulness of polyhistidine tails in affinity precipitation of recombinant proteins using metal chelates. The use of precipitation instead of chromatographic procedures has several advantages. The process can be made inexpensive and it is easy to scale up. Furthermore, the limitations frequently encountered using chromatography such as column fouling and nonspe- cific binding to the gel can be avoided.

In this study, we have used affinity tails composed of four or five histidine residues. Longer tails frequently reduce the level of gene expression, i.e., the amount of protein obtained after a microbial fermentation can be decreased severely. On the other hand, the binding between the tail and the chelated metal ion is often stronger using longer polyhistidine tails. This increase in apparent affinity is most likely due to a higher proba- bility of the chelated metal ion to encounter two imidazole groups on the protein in proper orientation for binding. In most instances, the use of four or more histidine residues assures strong metal-protein interactions.

The introduction of polyhistidine tails facilitated the purification of galDH and LDH remarkably. By a single precipitation step using EGTA(Zn)2, the enzyme recovery was around 70% and the degree of purification was over 20-fold, which made this technique comparable in efficiency with conventional affinity chromatography. The more time-consuming chromatographic procedures could hence be substituted with affinity precipitation. GalDH is a dimeric protein while LDH is tetrameric, but no significant difference in precipitation behavior


of these proteins was observed. The monomeric protein A(his)5 molecules could not be precipitated because the bis-ligand EGTA(Zn)2 requires at least two binding sites on the protein for aggregation to occur. However, when EGTA(CU)~ was utilized, the histidine-modified protein A could be precipitated. Native protein A con- tains three histidine residues and one or more of these may contribute to the increased precipitation using copper as chelating agent. By inserting two metal binding sites on monomeric proteins, for instance, by inserting polyhistidine tails both in the amino- and the carboxy- terminal ends, it is reasonable to assume that precipitation can be made effective for this group of proteins as well.

To achieve a high selectivity in the precipitation step, it is desirable that the affinity tail can bind weak metal chelates such as EGTA(Zn)2. Stronger chelates, such as EGTA(CU)~, may decrease selectivity; that is, proteins with naturally occurring metal binding properties will also start to precipitate. In addition, the binding between the metal chelate and the affinity tail must not denature the protein because the precipitate needs to be dissolved with preserved tertiary structure and enzyme activity. In accordance with this reasoning EGTA(Cu)2 proved less useful than EGTA(Zn)2. For instance, pre- cipitates of protein A(his)5 were difficult to dissolve.

During the formation of protein aggregates both in- tra- and intermolecular interactions may occur in the crosslinked protein aggregates, indicating that the length of the spacer in the bis-ligand may be critical for precipitation. The chelating agent, EGTA, proved to be effective although it is possible that a longer spacer region between the coordination sites for the metal ion may improve the precipitation efficiency. Another im- portant feature to keep in mind is the affinity between the metal ion and the chelating agent. Free metal ions can reduce the precipitation efficiency but, more impor- tantly free metal ions are common inhibitors of many proteins. This was probably the main reason for the low recovered activity of p-glu. The concentration of the metal chelate in the precipitation step was also critical. Addition of EGTA(Zn)2 above 3 mM resulted in reduced purity.

The affinity tails employed for protein precipitation also proved valuable for site-specific and reversible immobilization of proteins, particularly in the case of LDH(his)4. All enzymes were thus enzymatically active when adsorbed on chelating Sepharose. In most cases, the activity could be better retained if zinc instead of copper was used as metal ligand. However, the long- term stability was improved using copper. A plausible reason for this can be found in the multiple point attach- ment obtained between the protein and the gel using the latter metal ion, since the protein in many instances is able to bind to the chelated metal not only with the affinity tail but also with other residues in the native protein structure.

CONCLUSION

Metal affinity precipitation can be used as a simple and efficient method for purification of recombinant proteins. The procedure is applicable to oligomeric proteins, but may also be useful for monomeric if two or more metal binding regions are introduced on the poly- peptide chain. The introduction of polyhistidine tails can also be used for site-specific protein immobilization. This is particularly attractive for proteins which are sen- sitive to such chemical modifications.

The project was supported by grants from the Swedish Coun- cil for Forestry and Agricultural Research (SJFR) and the Swedish National Science Research Foundation (NFR).

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affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

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