©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Histidine Patch Thioredoxins
MUTANT FORMS OF THIOREDOXIN WITH METAL CHELATING AFFINITY THAT PROVIDE FOR CONVENIENT PURIFICATIONS OF THIOREDOXIN FUSION PROTEINS (*)

(Received for publication, November 8, 1995; and in revised form, December 7, 1995)

Zhijian Lu Elizabeth A. DiBlasio-Smith Kathleen L. Grant (§) Nicholas W. Warne Edward R. LaVallie Lisa A. Collins-Racie Maximillian T. Follettie Mark J. Williamson John M. McCoy (¶)

From the From Genetics Institute, Inc., Cambridge, Massachusetts 02140

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and -11 expressed in E. coli as soluble thioredoxin fusion proteins.


INTRODUCTION

In recent years the advance of recombinant protein production technology has contributed to a growth in our understanding of protein biochemistry and protein structure/function and has enabled the expanded use of recombinant gene products as therapeutic agents. In many instances the host cell chosen for recombinant gene expression is the bacterium Escherichia coli based on this organism's convenient culture characteristics, well developed gene expression systems, and ability to deliver high recombinant protein yields. However, recombinant proteins produced in E. coli often accumulate as insoluble cellular aggregates termed ``inclusion bodies.'' Although these inclusion bodies provide opportunities for easy initial purifications, this advantage is more than offset by the requirement for developing in vitro refolding protocols, an empirical process that is not always successful.

Inclusion body formation in E. coli can often be avoided by producing recombinant proteins as fusions, and a number of gene fusion expression systems that have high success rates in this regard are in common use(1, 2, 3, 4) . Moreover, correctly folded heterologous proteins can often be successfully isolated from fusion proteins following specific proteolysis(5, 6, 7) . Although the production of a soluble fusion protein eliminates the need for in vitro refolding, the easy initial purification step afforded by inclusion bodies is lost. To compensate for this, many fusion expression systems incorporate a specific affinity interaction that can be used as a ``purification handle.'' For example the maltose-binding protein fusion system takes advantage of the affinity between maltose-binding protein and amylose resins(2) , and the glutathione S-transferase system exploits the specific interaction of glutathione S-transferase with glutathione(1) . In addition, short peptide ``affinity tags'' grafted onto recombinant proteins, such as ``FLAG tag''(8) , ``Strep tag'' (9) or ``His6 tag''(10) , have been employed to aid in purifications.

Previously, we have described an E. coli gene fusion expression system that uses E. coli thioredoxin, an 11.7-kDa cytoplasmic protein, as a fusion partner(3) . This system can produce many mammalian proteins at high yields as soluble, functional thioredoxin fusions. Although thioredoxin fusion proteins can be readily purified by classical ion exchange chromatographic methods, we wanted to devise a convenient, generic, high-throughput affinity purification step for the thioredoxin fusion system. Immobilized metal ion affinity chromatography (IMAC)(^1)(11) possesses a number of advantages for recombinant fusion protein purifications: column matrices are cheap and readily available, certain proteins can bind with high specificity and good capacity, and although the buffers used are generally mild, binding is tolerant to a wide variety of solvent conditions, including the presence of detergents or denaturants such as urea(12) . Currently IMAC is most commonly used for recombinant protein purification in conjunction with a His6 affinity tag(10) , in which six histidine residues are attached to either the NH(2) or COOH terminus of the protein of interest. This has proven to be a particularly effective way to confer on a recombinant protein the ability to chelate divalent metal ions and thus the capacity to bind to IMAC resins. However, there are two potential problems with the His6 tag approach: 5` gene extensions can compromise overall gene expression in E. coli at the level of translation initiation, and COOH-terminal peptide tags are difficult to completely remove, because specificity determinants for most proteases lie on the NH(2)-terminal side of the scissile bond. We sought to take advantage of IMAC purifications for thioredoxin fusion protein purifications but in a manner that would avoid the potential problems of the His6 tag approach. In this paper, we describe the engineering of a metal affinity site on the surface of the thioredoxin molecule. We show that a metal ion binding capacity can be conferred on thioredoxin by changing two or three surface-exposed residues to histidine and that these mutant forms of thioredoxin (termed ``histidine patch'' or His patch thioredoxins) retain the wild-type molecule's ability to act as an effective fusion partner. We also show that fusion proteins containing histidine mutant thioredoxins can be recovered from crude bacterial lysates in a single IMAC step by columns charged with nickel ions. Furthermore we demonstrate that these fusions can be cleaved efficiently by recombinant enterokinase and illustrate the utility of the method by the production of highly purified recombinant human interleukin-11 and interleukin-8.


EXPERIMENTAL PROCEDURES

Materials

Iminodiacetic acid (IDA)-Sepharose was from Pharmacia Biotech Inc. and Ni-nitrilotriacetic acid (NTA) resin was from QIAGEN (Chatsworth, CA). QAE ion exchange resin was from TosoHaas (Montgomeryville, PA), and Sephadex G-75 resin was from Sigma. Enterokinase light chain was expressed in CHO cells and purified as described(6) .

Plasmids, Bacterial Strains, and Gene Expression Procedures

To produce the various histidine patch forms of thioredoxin, an oligonucleotide cassette mutagenesis procedure was performed on the wild-type E. coli thioredoxin gene (trxA) cloned in the expression vector, pALtrxA-781(3) . To construct His patch thioredoxin gene fusions, genes of interest were fused in-frame to the 3` end of the appropriate version of His patch thioredoxin using a spacer encoding the sequence GSGSGDDDDK. Fusion genes, under the transcriptional control of the bacteriophage pL promoter, were expressed in E. coli strain GI724 (3) or GI934, (^2)a strain that is isogenic with GI724 except for specific deletions in two E. coli proteases genes, ompT and ompP(13) . Briefly, an overnight culture of plasmid-containing bacteria was used to inoculate IMC/Amp medium (M9 medium containing 0.2% casamino acids, 0.5% glucose, 1 mM MgSO(4), and 100 µg/ml ampicillin) to an A of 0.05. The culture was grown at 30 °C until the A reached 0.5, then L-tryptophan was added to a concentration of 100 µg/ml, and the culture temperature shifted to 37 °C. 4 h following tryptophan addition, the cells were harvested by centrifugation and stored at -80 °C until use.

Computer Modeling of Histidine Patch Thioredoxins

The amino acid sequence of the hp2TrxA mutant was submitted electronically to the GLAXO Institute for Molecular Biology, Geneva, Switzerland (http:/www/expasy.hcuge.ch/swissmod) for computer modeling using the algorithm described by Peitsch and Jongeneel(14) . The ribbon diagrams of protein structures were drawn with the program MOLSCRIPT(15) .

Purification of Thioredoxin and His Patch Thioredoxin Mutants

E. coli cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, containing 1 mMp-aminobenzamidine and 1 mM phenylmethylsulfonyl fluoride) and lysed in a microfluidizer (Microfluidics, Newton, MA). As an alternative to cell lysis, thioredoxin and hpTrxA mutants were sometimes released from E. coli by an osmotic shock procedure(3) . Lysates were clarified by ultracentrifugation at 35,000 rpm in a Beckman Ti-50 rotor for 30 min and loaded onto QAE columns. After washing extensively with buffer (25 mM Tris-HCl, pH 7.5), bound materials were eluted with a linear gradient of 0-500 mM NaCl. Fractions were analyzed by SDS-PAGE, and those containing wild-type or mutant thioredoxins were concentrated in a vacuum-dialysis apparatus (Spectrum, Los Angeles, CA) using membranes with a molecular weight cut-off of 5000 daltons and a buffer comprising 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl. The concentrated thioredoxin samples were further purified by Sephadex G-75 gel filtration chromatography with the same buffer, and pooled fractions analyzed by Tricine SDS-PAGE(16) . Thioredoxin samples purified by QAE and G-75 chromatography were analyzed for binding to Ni-IDA and Ni-NTA columns (bed volume, 8 ml) on a Waters 650 high performance liquid chromatography system.

Purification of IL-11 and IL-8

hp2TrxA/IL-11 and hpTrxA/IL-8 fusions were produced in strain GI934. Cells were lysed as described above, with NaCl and imidazole added to the lysates to 500 mM and 2 mM, respectively. The lysate was allowed to sit on ice for 30 min before cell debris was removed by ultracentrifugation at 35,000 rpm in a Beckman Ti-50 rotor for 30 min. The clarified supernatant was then loaded onto a Ni-IDA column at a flow rate of 20% of the resin volume/minute. The column was subsequently washed thoroughly with buffer A (25 mM Tris-HCl, pH 7.5, 2 mM imidazole, 200 mM NaCl) and eluted with a linear gradient of buffer B (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 100 mM imidazole). Fractions containing fusion protein were pooled and dialyzed extensively against enterokinase digestion buffer (10 mM Tris-HCl, pH 7.5, and 75 mM NaCl). Enterokinase cleavage was carried out by adding enterokinase light chain to the fusion protein at a ratio of 1:15,000 (w/w) and incubating overnight at 37 °C. The digest was then reapplied over the original Ni-IDA column re-equilibrated with buffer A. Flow-through fractions (or in the case of IL-11, the fractions resulting from a 25 mM Tris-HCl, pH 7.0, 200 mM NaCl, 2 mM imidazole wash) were analyzed by Tricine SDS-PAGE to identify cytokine-containing fractions.

Thermostability Measurements

Thermal denaturation experiments were performed using a SLM/Aminco 8000C fluorescence spectrophotometer. The temperature of the cuvette was maintained with a Neslab RTE-110 programmable water bath. Sample temperature was recorded directly using a small thermocouple (Physitemp MT-23/5). Protein samples, dialyzed against 50 mM sodium phosphate, pH 7.00 and diluted to 1.6 mg/ml with the same buffer, were excited at 292 nm, and emission was monitored at 340 nm. Temperature was increased from 21 to 100 °C over a period of 83 min. Determination of the melting temperature (T(m)) was performed by geometrically measuring the midpoint of the curve between the extrapolated predenaturation region and postdenaturation region or by first derivative analysis.


RESULTS

Construction, Expression, and Purification of His Patch Thioredoxin Mutants

Visual inspection of the crystal structure of E. coli thioredoxin (17) reveals a cluster of residues (Ser-1, Glu-30, and Gln-62) in the vicinity of the molecule's single histidine residue at position 6, whose side chains are positioned on the surface of the molecule (Fig. 1A). These side chains make no significant contacts with the side chains of neighboring residues, and furthermore an examination of their crystallographic R-factors indicates a moderate degree of disorder, which suggests that they possess a reasonable degree of conformational freedom. These residues can be positioned within an imaginary sphere with a radius consistent with the coordination of an average sized transition metal ion such as copper, cobalt, or nickel, assuming the presence within the sphere of the appropriate coordinating ligands (Fig. 1A). To provide these ligands we systematically changed, either individually or in various combinations, residues Ser-1, Glu-30, and Gln-62 to histidine, a residue known to bind to metal ions at physiological pH. In some instances the His-6 residue was changed to asparagine and Asp-26 to alanine (Table 1). All of the mutated thioredoxin genes, when expressed in E. coli strain GI724 according to the protocol described above, were found to produce soluble mutant proteins at levels comparable with that of the wild-type thioredoxin (data not shown), confirming our suspicion that the residues targeted for mutagenesis were not significantly involved in the folding or final stability of the molecule.


Figure 1: A, structure of wild-type thioredoxin. Shown in the figure is a ribbon representation of the thioredoxin alpha-carbon backbone as determined by x-ray crystallography. The arrow indicates the COOH-terminal carboxyl group where the fusion linker is attached. The side chains of five residues are shown in the figure: Ser-1, His-6, Asp-26, Glu-30, and Gln-62. In various thioredoxin mutants Ser-1, Glu-30, and Gln-62 were changed to histidine, His-6 was changed to asparagine, and Asp-26 was changed to alanine, either individually or in combination, resulting in the different thioredoxin mutants listed in Table 1. B, a molecular model of hp2TrxA in which histidine side chains replaced the native glutamate and glutamine side chains at positions 30 and 62, respectively. The ND1 nitrogen of His-30 and the NE2 nitrogen of His-62, the proposed metal chelating groups, are indicated by arrowheads. The drawings were made with the program MOLSCRIPT(15) .





All of the mutants listed in Table 1were tested qualitatively for their ability to bind to IDA resin charged with either Cu or Ni ions. Small scale samples of dialyzed wild-type and mutant thioredoxins purified by QAE chromatography were loaded onto small cartridges containing the metal ion-charged resin, which were then washed with a solution of 25 mM Tris-HCl, pH 8, containing 100 mM NaCl and 2 mM imidazole, followed by the same buffer containing 100 mM imidazole. Washings and eluates were analyzed by SDS-PAGE, with the results shown in Table 1. Wild-type thioredoxin and all of the two histidine-containing mutants, with the exception of H30,62TrxA, failed to bind to IDA resin charged with either Cu or Ni. Mutant H30,62TrxA (two-histidine mutant), hp2TrxA (three-histidine mutant), and hpTrxA (four-histidine mutant) were all found to bind to both Ni-IDA and Cu-IDA resins, and all could be specifically eluted with a buffer containing 100 mM imidazole. The above observations suggested strongly that H30,62TrxA, hp2TrxA, and hpTrxA interacted with metal ions via coordination to the introduced histidine residues; furthermore the interactions did not appear to be ionic in character because they were stable to concentrations of sodium chloride of up to 2 M (data not shown). The results indicated that the key residues capable of coordinating metal ions were His-30 and His-62.

Samples of the three metal ion-binding His patch thioredoxins were purified for further analysis by QAE ion exchange, concentration dialysis, and G-75 size exclusion chromatography. Fig. 2shows samples of these preparations analyzed by SDS-PAGE on a 12% Tricine gel(16) . The proteins were estimated to be >98% homogeneous.


Figure 2: SDS-PAGE analysis of the purified thioredoxins. Wild-type and His patch thioredoxins were purified by QAE and G-75 chromatography (see ``Experimental Procedures'' for details). The purified proteins were analyzed by 12% Tricine SDS-PAGE and visualized by Coomassie Blue staining. Lane 1, TrxA; lane 2, H30,62TrxA; lane 3, hp2TrxA; lane 4, hpTrxA; lane 5, D26A; lane 6, hp2D26ATrxA; and lane 7, hpD26ATrxA.



Behavior of His Patch Thioredoxins on IMAC Columns

To further characterize interactions between the His patch thioredoxins and metal chelating resins, purified samples of H30,62TrxA, hp2TrxA, hpTrxA, and wild-type thioredoxin were bound to Ni-IDA and Ni-NTA columns and eluted with linear imidazole gradients. Retention times for wild-type thioredoxin were consistent with elution in the void volume for both Ni-IDA (Fig. 3A) and Ni-NTA (Fig. 3B) columns, indicating that the protein did not interact with either resin. In contrast, mutants H30,62TrxA and hp2TrxA bound tightly to both resins, exhibiting identical retention times (15.4 min on Ni-IDA and 13.3 min on Ni-NTA). Ni ions chelated to NTA resin have two coordination sites available for binding to protein, whereas Ni ions chelated to IDA resin have three available sites. Because H30,62TrxA and hp2TrxA bind to each resin with the same apparent affinity, it is unlikely that the histidine at position 6 of hp2TrxA participates in binding. This conclusion is supported by the observation that all mutant forms of thioredoxin containing only his-6 and one other histidine did not bind to either resin.


Figure 3: Retention behavior of wild-type and mutant thioredoxins on Ni-IDA and Ni-NTA columns. Thioredoxin samples purified by QAE and G-75 chromatography were analyzed by Ni-IDA (A) and Ni-NTA (B) columns (8 ml of resin bed). Buffer A, 25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM imidazole. Buffer B, 25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 100 mM imidazole. The flow rate was 3 ml/min. On both Ni-IDA and Ni-NTA columns, the wild-type thioredoxin did not bind to the metal chelating resins, whereas H30,62TrxA and hp2TrxA bound and displayed identical retention times. hpTrxA, which has one more histidine residue than hp2TrxA, showed longer retention times and a broader elution profile on both columns.



hpTrxA, which has an additional histidine residue at position 1, exhibited longer retention times (17.4 min on Ni-IDA and 14.5 min on Ni-NTA) than hp2TrxA and a broader elution profile on both columns, hinting that there may be an additional mode of interaction between this protein and IMAC resins that includes its amino-terminal histidine residue. In general it appears that the His patch thioredoxins have retention times 10-20% greater on Ni-IDA columns than on Ni-NTA resins, probably a result of differences in functional group densities between the two resins(11, 18) .

His Patch Thioredoxin Fusions

To test the ability of His patch thioredoxins to act as effective fusion partners and to serve as models for the development of a simplified generic purification scheme for His patch thioredoxin fusion proteins, fusions to two human cytokines were constructed. Mature human IL-11 was fused to the COOH terminus of hp2TrxA and mature human IL-8 (77-amino acid form) to the COOH terminus of hpTrxA by a linker peptide consisting of GSGSG and the enterokinase recognition sequence DDDDK. Because the enterokinase cleaves after the lysine residue, no extra residues derived from the linker remain on the cytokines following enterokinase cleavage. Expression of these fusions was performed in the protease deleted strain GI934 (DeltaompT,DeltaompP). After cell lysis, NaCl and imidazole were added to 500 mM and 2 mM, respectively, and the cell debris was removed by ultracentrifugation. The resulting supernatants (Fig. 4, A and B, lanes 1) show that both His patch fusions were produced as soluble proteins, although each was present at a different overall level. Following passage of the clarified lysates over Ni-IDA columns, both fusions were efficiently adsorbed, with little remaining in the flow-through fractions (Fig. 4, A and B, lanes 2). The capacity of the columns for binding His patch thioredoxin fusions was high. In a separate experiment (data not shown), we determined that up to 6.5 mg of hp2TrxA/IL-11 fusion protein could be bound per ml of Ni-IDA resin. Following extensive washing the fusions were cleanly eluted with imidazole. Fractions containing hpTrxA fusion proteins (Fig. 4, A and B, lanes 3) were substantially purified, although not to homogeneity. After buffer exchange, fusions were cleaved by recombinant enterokinase light chain (Fig. 4, A and B, lanes 4) and the digests were reapplied to the Ni-IDA columns. As can be seen (Fig. 4, A and B, lanes 5), mature, highly purified preparations of human IL-11 and IL-8 were recovered from the flow-through. For IL-11, the protein was homogeneous as judged from overloaded Coomassie Blue-stained gels (Fig. 4A, lane 5). Although the IL-8 preparation exhibited a multiple band pattern by Coomassie Blue staining (Fig. 4B, lane 5), all the bands were found to cross-react with a monoclonal anti-IL8 antibody by Western blot analysis (data not shown), indicating that extraneous bands were due to either aggregation or proteolytic degradation of IL-8 and not to contaminating E. coli proteins.


Figure 4: Purification of human cytokines IL-11 and IL-8. hp2TrxA/IL-11 and hpTrxA/IL-8 fusions were expressed in E. coli strain GI934, and the entire purification procedure was described under ``Experimental Procedures.'' The samples were analyzed by 12% Tricine SDS-PAGE and visualized by Coomassie Blue staining. A, purification of IL-11. B, purification of IL-8. Lanes 1, Cell lysate supernatants containing the corresponding His patch thioredoxin fusions; lanes 2, flow-throughs from Ni-IDA columns; lanes 3, pools of fractions containing the fusions eluted from the column with imidazole (after dialysis); lanes 4, enterokinase digestions of the fraction pools; lanes 5, flow-through (or pH 7 wash for IL-11) of the enterokinase digests when re-applied to Ni-IDA columns; and lanes 6, components in the enterokinase digests adsorbed on the Ni-IDA matrices and eluted with 100 mM imidazole buffer.



Thermostability of His Patch Thioredoxins

Because it is likely that the ability of thioredoxin to act as a successful fusion partner is intimately linked to its structural stability, we were concerned that the molecule's capacity to serve in a fusion partner role might be compromised by the introduction of multiple mutations, even mutations effecting only surface residues. As a gauge of structural integrity we assessed the thermostabilities of His patch thioredoxins using temperature scanning intrinsic fluorescence measurements(19) . The results are shown in Fig. 5and Table 2. As can be seen, the melting temperature of wild-type thioredoxin was determined as 86.0 °C, compared with reported literature values for wild-type thioredoxin lying between 85.82 and 86.01 °C under the same solvent conditions(20) . The melting temperatures of H30,62TrxA, hp2TrxA, and hpTrxA were 78.2, 79.9, and 78.1 °C respectively, all about 6-8 °C lower than that of the wild-type molecule but still remarkably high, indicating maintenance of a stable structural fold.


Figure 5: Thermal denaturation of the wild-type and mutant thioredoxins monitored by tryptophan fluorescence. Protein samples (see ``Experimental Procedures'' for preparation) were excited at 292 nm, and emission was monitored at 340 nm. The temperature of the sample solution was measured with a small thermocouple and plotted against the fluorescence intensity. The T(m) for all the samples were determined by geometrically measuring the midpoint of the curve between the extrapolated predenaturation region and postdenaturation region, except for D26A, which is estimated by first derivative analysis. , wild type; circle, H30,62TrxA; box, hp2TrxA; , hpTrxA; bullet, D26A; , hp2D26ATrxA; up triangle, hpD26ATrxA.





It had previously been reported that a thioredoxin mutant, D26A, was more stable to denaturation by guanidine HCl than was wild-type thioredoxin(21) . We found D26A to be very stable as determined by temperature scanning fluorescence measurements. In fact we did not see complete denaturation of this mutant up to 99.3 °C (Fig. 5) and could estimate its melting temperature to be 96.8 °C only from a first derivative calculation. This melting temperature was more than 10 °C higher than that of wild-type thioredoxin. We therefore attempted to counteract the negative effects of the multiple histidine changes on thioredoxin melting temperature by incorporating the D26A mutation into His patch thioredoxins. Introduction of the D26A change into hpTrxA and hp2TrxA indeed increased the melting temperatures of both these molecules by 8.7 °C (Table 2).


DISCUSSION

Thioredoxin gene fusions have proven themselves to be useful for producing heterologous proteins in E. coli in a soluble form (3, 22, 23) . To provide for easier purifications of thioredoxin fusion proteins, we investigated the possibility of incorporating a metal ion binding site onto thioredoxin's molecular surface, with a view to providing specific affinity toward IMAC resins. By examining the crystal structure of thioredoxin (17) we found four surface residues, Ser-1, His-6, Glu-30, and Gln-62, with a geometry which theoretically would allow the imidazole side chains of histidine residues placed at these positions to coordinate the sp^3d^2 orbitals of medium sized transition metal ions under physiological conditions. These four residues are not adjacent in the thioredoxin primary sequence; instead they come together as a result of thioredoxin's tertiary fold to form a patch on the molecular surface. Mutant forms of thioredoxin with histidine substitutions at these positions were thus called histidine patch thioredoxins.

We systematically mutagenized Ser-1, Glu-30, and Gln-62 to histidine in order to ascertain the minimal requirements for metal binding. In some instances we also changed the native histidine at position 6 to asparagine. In column binding experiments using Ni or Cu ions immobilized onto IDA-Sepharose, the various His patch thioredoxins demonstrated distinctly different properties. It was clear that binding only occurred when histidine residues were present at both positions 30 and 62, as was the case for the H30,62TrxA, hp2TrxA, and hpTrxA mutants. Moreover H30,62TrxA and hp2TrxA exhibited identical retention behavior in column elution experiments using Ni-NTA and Ni-IDA resins. Four of nickel's six coordination sites are occupied by matrix ligand groups on NTA resin, with only two sites remaining free for protein binding(18) . With IDA resins three metal ion coordination sites remain available, yet for both matrices the strength of binding interactions observed for the H30,62TrxA and hp2TrxA mutants were identical. This argued against any involvement in binding for the native His-6 residue of hp2TrxA. It thus appears that histidine residues placed at positions 30 and 62 of thioredoxin were both necessary and sufficient for metal ion binding.

It has been demonstrated that two vicinal histidines (His-84 and His-85) in rat glutathione transferase 3-3 can bind to Ni-IDA resin, with the distance separating the chelating nitrogen ligands on the two histidine residues measured to be between 7.5 and 8.0 Å(24) . Although we did not perform structural analyses on the His patch thioredoxins, we did generate a model of hp2TrxA using the method of Peitsch and Jongeneel (14) and used it to measure the distances between the potential coordinating nitrogen atoms of histidine residues. As illustrated in Fig. 1B, the distance between the ND1 nitrogen atom of His-30 and the NE2 nitrogen atom of His-62 in this model is 7.5 Å, consistent both with the measured distance for the two nickel ion chelating histidine nitrogens in rat glutathione transferase and with our experimental results implicating His-30 and His-62 in nickel ion binding. In the model no other pair of side chain nitrogens originating from separate histidines comes closer than 7.5 Å.

In order to understand the broad elution profile of hpTrxA on Ni-NTA and Ni-IDA columns, we tried to identify other possible chelation modes. We measured the distances between the beta-carbons of residues Ser-1, His-6, Glu-30, and Gln-62 in wild-type thioredoxin (17) and found the beta-carbon distance between Ser-1 and His-6 (9.31 Å) to be similar to that measured between Glu-30 and Gln-62 (9.46 Å). Based on these data and on the fact that H30,62TrxA is able to bind to metal columns, we postulated that the histidine pair in mutant H1,6TrxA might also be able to chelate immobilized metal ions. However, this proved not to be the case experimentally. One possible explanation for this is that the ability of H1,6TrxA to bind metal ions is compromised by conformational disorder at the thioredoxin amino terminus, disorder that is indeed suggested in the solution structure of thioredoxin(25) . Although H1,6TrxA fails to bind to metal ions as judged by its behavior on IMAC columns, the reproducible difference in column elution profiles between hp2TrxA and hpTrxA suggests that the histidine at position 1 in hptrxA may in fact provide weak additional metal binding affinity. This hypothesis can be tested by a more sensitive measurement of the affinities for immobilized nickel of all the two-histidine thioredoxin mutants.

It is interesting to note that His patch thioredoxin mutants apparently bind to immobilized copper (II) ion as well they bind to nickel (II), even though the two metal ions have different sizes and preferred coordination numbers(26) . This may suggest that liganding groups in the mutants possess sufficient conformational flexibility to accommodate binding to different metals.

Ni-IDA and Ni-NTA resins have high reported protein binding capacities due to high ligand densities(11, 18) . We found that capacities for His patch thioredoxins were greatly reduced unless E. coli lysates containing His patch fusion proteins had previously been adjusted to high ionic strength and clarified by ultracentrifugation. The plausible causes for the apparent interference to the binding of fusion proteins include the ionic interaction of E. coli proteins to resin functional groups and/or the binding of E. coli outer membrane debris to the immobilized metal ions. Such interactions would occupy column binding sites and, especially in the case of membrane debris, mask a large column surface area. The clarification procedure combined with elevating ionic strength of the lysates effectively preserved high binding capacity for His patch thioredoxin fusions on Ni-IDA resin. Alternatively, a simple QAE step, as was used for the His patch thioredoxin mutants in this work, can be incorporated into the purification scheme for His patch thioredoxin fusion proteins, particularly those fusion genes expressed at low level.

Although a number of thioredoxin fusions are biologically active and can be used in bioassays directly(3, 23) , it is usually desirable to separate the protein of interest from the fusion partner. One great advantage of a fusion partner that carries an affinity purification handle is the ability to run the same column purification step twice, both prior to and following fusion protein cleavage. In this way contaminants that bind and elute with the fusion protein on the first column bind again along with the cleaved fusion partner on the second column. The protein of interest can then be recovered in a highly purified form in the flow-through fractions of the second column. We successfully demonstrated this purification strategy by utilizing His patch thioredoxin fusions to purify essentially homogeneous human IL-11 and IL-8.

A distinguishing feature of thioredoxin is its extraordinary thermostability. We wondered what changes in thermostability the various histidine mutations might cause and what deleterious effects these changes might have on thioredoxin's ability to act as a good fusion partner. We therefore studied the melting temperature of representative His patch thioredoxins by temperature scanning intrinsic fluorescence measurements. All of the His patch thioredoxins tested had melting temperatures 6-8 °C lower than that of the wild type. Despite these changes, all of the His patch mutants had T(m) values close to 80 °C, thus all are extremely stable molecules. Because all the newly introduced histidines in the His patch mutants replaced hydrophilic surface residues, it is possible that changes in local surface charge distribution may have been a direct cause of the T(m) decreases. For example, hp2TrxA would have a net decrease of one negative charge in the patch region at the test pH, and it is possible that this change may have led to a realignment of surface charge distribution, which in turn may have distorted the main chain backbone enough to destabilize the protein.

Aspartate residue 26 of thioredoxin serves an important functional role (21, 27) and is buried in the hydrophobic core of the protein(17) . It has been reported that replacing this residue with alanine leads to an increase in the concentration of guanidine hydrochloride required for denaturation(21) , thus we attempted to stabilize His patch thioredoxin mutants by incorporating the D26A mutation. In heat denaturation experiments, mutant D26A demonstrated a T(m) of 96.8 °C, well above that of wild-type thioredoxin. In addition, introduction of the D26A mutation into His patch thioredoxins yielded significant improvements in protein thermostability. We are currently investigating the performance of hp2D26ATrxA as a fusion partner.

Even though we found that engineering a metal binding site onto thioredoxin adversely affected its thermostability, we also found that His patch thioredoxins retained the ability to form soluble fusions. Soluble production levels of hpTrxA fusions to IL-11, IL-8, and several other proteins (data not shown) were as good as the corresponding wild-type thioredoxin fusions. However a hpTrxA/IL-6 fusion tended to appear in the ``insoluble'' cellular fraction, whereas the wild-type thioredoxin/IL-6 fusion was fully soluble. We determined that the cause was an enhanced affinity of this fusion for E. coli membranes, perhaps via interactions to metal ions bound to outer membrane lipopolysaccharide. Washing the unlysed cells with a 1% sarcosyl solution and subsequent sarcosyl removal prior to cell lysis effectively prevented the appearance of hpTrxA/IL-6 in the insoluble fraction.

In conclusion we have generated and characterized mutant forms of E. coli thioredoxin in this work, called His patch thioredoxins, that possess an engineered metal ion binding site on their molecular surface. The production and purification of two human cytokines illustrates the effectiveness of these His patch thioredoxins as fusion partners, and highlights the utility of an IMAC purification handle.


FOOTNOTES

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Present address: BASF Bioresearch Corporation, 100 Research Dr., Worcester, MA 01605-4314.

To whom correspondence should be addressed: Genetics Inst., Inc., 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-498-8225; Fax: 617-498-8878; :jmccoy{at}genetics.com.

(^1)
The abbreviations used are: IMAC, immobilized metal ion affinity chromatography; IDA, iminodiacetic acid; NTA, nitrilotriacetic acid; TrxA, thioredoxin; IL-8, interleukin-8; IL-11, interleukin-11; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

(^2)
E. A. DiBlasio-Smith and J. M. McCoy, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Roger Brent, Neil Wolfman, Jeff Deetz, and Neil Schauer for helpful discussions during the course of this work, Hsiang-Ai Yu and Will Somers for computer graphics, and the Genetics Institute DNA synthesis and DNA sequencing groups for their technical support.


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