(Received for publication, November 8, 1995; and in revised form, December 7, 1995)
From the
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.
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)()(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
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
-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.
Figure 1:
A, structure of
wild-type thioredoxin. Shown in the figure is a ribbon representation
of the thioredoxin -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.
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) .
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.
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 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;
, H30,62TrxA;
, hp2TrxA;
, hpTrxA;
,
D26A;
, hp2D26ATrxA;
,
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).
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 spd
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
-carbons of residues Ser-1, His-6, Glu-30, and Gln-62
in wild-type thioredoxin (17) and found the
-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 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
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 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.