competitive elution of protein a fusion proteins allows specific recovery under mild conditions

Eur. J. Biochem. 224, 103-108 (1994) 0 FEBS 1994

Competitive elution of protein A fusion proteins allows specific recovery under mild conditions Joakim NILSSON, Peter NILSSON, Ylva WILLIAMS, Lena PETTERSSON, Mathias UHLEN and Per-Ake NYGREN Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden

(Received April 61May 31, 1994) - EJB 94 048313

A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG-binding staphylococcal protein A fragment (Z) from an IgG-affinity matrix by a divalent fragment fused to a serum-albumin-binding region derived from streptococcal protein G. Using real-time interaction analysis, the binding (KJ to polyclonal human IgG was found to be 3.3 ( -+0.4)X10'M-' for divalent ZZ and 2.0 ( 2 0.1)X lo7 M-' for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)-affinity column, leaving only the target fusion protein in the flow-through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG- affinity chromatography using mild conditions during both binding and elution.

Gene fusion has become an important tool in molecular biology with a wide variety of applications such as facilitated protein recovery [l -31, solubilization of proteins during in vitro refolding [4, 51, selection systems [6, 71, vaccine development [8] and assembly of chimeric protein for multiple functions [9-111. The use of an affinity tail as fusion partner to a target protein makes it possible to immobilize the protein onto a solid carrier and to purify it efficiently by affinity chromatography. A number of affinity fusion systems have been described based on different classes of interactions, such as, protein-protein, protein A-IgG [12, 131, protein G- human serum albumin (HSA) [14], antibody-antigen [15], strep-tag- streptavidin [ 161 ; enzyme-substrate, P-galactosi- dase-p-aminophenyl-thio-P-D-galactoside [17], glutathione- S-transferase-glutathione [18] ; protein-carbohydrate, maltose-binding-protein - amylose [ 19 ] ; protein-DNA, lac-re- pressor-lac-operator [20]; histidines-metals, immobilized- metal affinity chromatography [21]. These systems differ in several aspects such as size of fusion partner, secretion com- petence, cost for affinity resins and bindinglelution conditions.

Generally, an ideal system should combine a strong and highly specific interaction between a secretable protein and the ligand to allow for efficient recovery with high yield and purity, together with the possibility of gentle elution. The well characterized system utilizing the interaction between protein A (or the 14-kDa divalent analogue, designated ZZ) and the Fc of IgG fulfills most of these criteria and has suc- cessfully been used for the purification of recombinant pro-

Correspondence to P.-A. Nygren, Department of Biochemistry and Biotechnology, Royal Institute of Technology, Teknikringen 34, S-100 44, Stockholm, Sweden

Abbreviations. HSA, human serum albumin ; RU, resonance units.

Enzyme. DNA polymerase (EC 2.7.7.7).

teins produced in different hosts, such as bacteria [22], yeast [23], insect cells [24] and Chinese hamster ovary (CHO) cells (Lind, P., unpublished results). However, the elution of the fusions by low pH used routinely, can for some products be destructive, leading to a biologically inactive product. Here, we describe an elution strategy based on competition, which combines the high performance of the protein A fusion system with physiological conditions throughout the complete purification procedure.

EXPERIMENTAL PROCEDURES

Bacterial strains and plasmid vectors Escherichia coli strain RRIdM15 cells [25] were used

both as bacterial PCR template and host for cloning and expression of the fusion proteins. Plasmids pUC9 [26], pRIT28 [27], pRIT24 [28] and pRIT28-STOP (this study) were used as vectors for cloning. Plasmid pRIT28 was used also for the enzyme activity assay. The E. coli expression vector pRIT44 is described in [29].

DNA constructions All recombinant DNA manipulations followed standard

procedures [30]. The gene fragment encoding the Klenow fragment (residues 324-928) of E. coli DNA polymerase I was isolated by PCR, directly on the genomic DNA from a bacterial colony, using oligonucleotides Klen-5, 5'-GGGAA- TTCCGTGATTTCTTATGACAACTAC-3, and Klen-3,5'-G- GGGATCCGTGCGCCTGATCCCAGTTTTCG-3', desig- ned according to the sequence published by Joyce et a1.[31]. The E. coli sample (RRIdM15) was lysed in 5 pl distilled water at 99°C for 5 min and transferred to a reaction tube containing 50 pl PCR buffer (0.2 mM dNTP, 50 mM KC1,

104

10 mM Tris/HCl, pH 8.3, 2 mM MgCL 0.1% Tween 20), 5 pmol of each primer and 1.0 U of Taq DNApolymerase (Boehringer Mannheim). The PCR program (93 "C, 1 .O min; 55 "C, 1 .O min ; 72 "C, 1 .O min) was repeated for 35 cycles in a PHC-I thermocycler (Techne Inc.). The PCR product was restricted with EcoRIand BamHI using the endonuclease sites introduced during the PCR and inserted into the multilinker of pUC9 previously restricted with the same enzymes. Positive clones were further analyzed by restriction fragment mapping of the Klenow gene [31]. After mapping, pUC9-Klenow was redigested with EcoRI and BamHI and the Klenow-encoding gene inserted into pRIT28-STOP, previously digested with the same enzymes. This plasmid was constructed from pRIT28 [28] by insertion of a linker sequence 5'-GGATCCTAAAAGCTT-3' between the BamHI and HindIII sites of the cloning linker of pRIT28, resulting in an in-frame termination codon (TAA) directly after the BamHI site. To construct an expression vector suitable for fusion of the Klenow fragment to a single Z domain, the expression vector pRIT44 [29] encoding a divalent IgG-binding ZZ protein under control of the E. coli trp promoter was digested with BgZII, and the vector part purified and reli- gated. The resulting vector, pRIT45, encodes a single Z domain still under the control of the trp promoter. Plasmid pRIT28-Klenow was restricted with EcoRI and HindIII and the Klenow gene fragment was inserted into the expression vector pRIT45, yielding pRIT5O encoding a tripartite fusion protein (Z-Klenow) consisting of the first eight amino acids of the Trp leader peptide, a synthetic monovalent IgG-binding domain Z, derived from staphylococcal protein A and the Klenow fragment of the E. coli DNA polymerase I. For this construct, the intracellular expression is under control of the trp promoter.

To construct the expression vector encoding the competitor ZZ-BB, plasmid pRIT24 was restricted with EcoRI and HindIII yielding a gene fragment encoding the semm-albumin-binding region BB of streptococcal protein G [14]. The BB fragment was inserted into the multilinker of pRIT44 previously digested with the same enzymes. The resulting plasmid pRIT49 thus encodes a tripartite fusion protein (ZZ- BB) consisting of the first eight amino acids of the Trp leader peptide, a divalent IgG-binding domain ZZ and the serum- albumin-binding region (BB) from streptococcal protein G.

Gene expression and protein purification E. coli RRIdM15 cells harbouring plasmids pRIT49 and

pRIT5O were grown overnight at 37°C in shaken flasks containing 20 ml Tryptic Soy Broth (Difco) supplemented with 7 g/l yeast extract (Difco), tetracycline (8 pg/ml) and ampi- cillin (100 pg/ml). The overnight cultures were diluted 25- fold into shaken flasks containing 1000 ml of the same media and grown at 37°C. Expression of the recombinant fusion proteins were induced at mid-log phase (A58o = 1) by the addition of P-indole acrylic acid (Sigma) to a final concentration of 25 pg/ml. Cells were harvested 3 h after induction, by centrifugation at approximately 5000 g for 5 min. The pelleted cells were resuspended in 50 ml washing buffer (50 mM Tris/HCl, pH 7.5, 0.15 M NaC1, 0.05% Tween 20). When resuspending cells producing the Z-Klenow fusion protein, the washing buffer was supplemented with 1 mM dithiothreitol. The resuspended cells were lysed by sonica- tion and pelleted by centrifugation at approximately 20000 g for 15 min. Supernatants containing Z-Klenow fusion were filtered (1.2 pm; Millipore) and stored at -20°C. Purifica-

tion of the ZZ-BB fusion protein was performed by affinity chromatography on HSA-Sepharose as previously described [14]. The Z and ZZ proteins used in the affinity constant determinations on BIAcoreTM (Pharmacia Biosensor AB) were expressed intracellularly from plasmids pRIT44 and pRIT45, respectively, and purified by IgG-affinity chromatography according to standard procedures [12, 131

Column-capacity measurement To determine the capacity of the IgG-Sepharose, a 0.9-

ml column was saturated using a bacterial lysate containing approximately 20 mg Z-Klenow fusion, washed and eluted as described above. The amount of eluted Z-Klenow fusion (9 mg) was used to calculate the molar excess used in the competitive elution experiment.

Competitive elution The samples were loaded at 0.5 ml/min on a Phannacia

HR 5/5 column containing 0.9 ml IgG-Sepharose, using an FPLC system (Pharmacia Biotech AB). For protein quality controls, production level and column-capacity measurements, the IgG-Sepharose column was washed with 20 column volumes of washing buffer, followed by 5 volumes of 10 mM ammonium acetate, pH 7.5. The Z-Klenow fusion was then eluted with 0.3 M acetic acid, pH 3.1, at 0.2 ml/ min. In the competitive elution experiment, the column was after loading washed with 20 column volumes of washing buffer (supplemented with 1 mM dithiothreitol), followed by 5 volumes of 150 mM ammonium acetate, pH 7.5, 1 mM dithiothreitol. The competitive elution was performed at a flow rate of 0.1 ml/min for 20 min with affinity-purified ZZ- BB fusion protein (9.5 mg/ml) in 150 mM ammonium acetate, pH 7.5, 1 mM dithiothreitol. Subsequently, the column was washed with 10 column volumes of 150 mM ammonium acetate, pH 7.5, 1 mM dithiothreitol. Fractions of 0.1 ml were collected starting at the injection of the competitor. All fractions, except those withdrawn for SDSIPAGE analysis (12 fractions), were pooled and loaded onto an HSA-Sepha- rose column, previously equilibrated with washing buffer (supplemented with 1 mM dithiothreitol). The column was subsequently washed with 25 ml washing buffer (supplemented with 1 mM dithiothreitol) and the total flow through (29.8 ml) collected. Samples for SDSPAGE analysis and enzyme activity were withdrawn and the rest of the solution was stored at 4°C. Proteins captured on the HSA-Sepharose column were eluted as described above and stored at - 20 "C.

Protein analysis Protein concentrations were determined by absorbance

measurements at 280 nm using the absorption coefficients 0.71 cm2/mg and 0.33 cm2/mg for the fusions Z-Klenow and ZZ-BB, respectively. SDSPAGE was performed on the PhastTM system (Pharmacia AB) and on homogeneous 12% slab gels (BioRad Inc) according to Laemmli [32]. The gels were stained with Coomassie Brilliant Blue R-250.

BIAcoreTM analysis The affinities for polyclonal human IgG (hIgG) (Phar-

macia AB) of monovalent (Z) and divalent (ZZ) proteins were determined using the BIAcoreTM (Pharmacia Biosensor AB, Sweden). The carboxylated dextran layer of a CM-5

105

sensor chip was activated using N-hydroxysuccinimide and N-ethyl-N’-(3-diethylaminopropyl)-carbodiimide chemistry according to the manufacturer. For immobilization, the hIgG was diluted in 10 mM sodium formate, pH 4.0, to a concentration of 100 nM and 20 pl was injected at 5 pVmin over the activated surface, resulting in approximately 6000 resonance units (RU) of immobilized hIgG. The Z and ZZ proteins were dissolved in NaCVHepes (10 mM Hepes, pH 7.4, 150 mM NaC1, 3.4 mM EDTA, 0.05% surfactant P-20) to 1600, 800, 400, 200, 100 and 50 nM, and 20 pl of each sample was injected (5 pVmin). The resulting sensorgrams from three independent complete runs were evaluated using the BIAlo- gueTM kinetics evaluation (Pharmacia Biosensor AB) soft- ware to calculate the mean kon and k,, values.

Enzyme assay The specific DNA-polymerase activity of Z-Klenow fu-

sion protein obtained from the competitive elution was compared to a commercial Klenow fragment (Boehringer Mann- heim) in a biotin-dUTP fill-in reaction of protruding ends of plasmid pRIT28 linearized with the endonuclease HindIII. The amounts of the enzymes were estimated from SDS/ PAGE (PhastTM system, Pharmacia AB). 10-pM samples were diluted (1 : 1, 1 : 5, 1 : 25, 1 : 125) in 10 mM TrisMCl, pH 7.5, 100 pg/ml BSA, 10 mM MgC1, and 1 mM dithiothreitol. Sample aliquots were subsequently mixed with approximately 500 ng linearized pRIT28 and nucleotides to yield the final assay buffer concentration, 10 mM TrisMCI, pH 7.5, 20 pM nucleotides (biotin-dUTP, dATP, dCTP and dGTP ; Boehringer Mannheim) ; 10 mM MgC12, 10 pg/ml BSA and 0.1 mM dithiothreitol. After incubation for 15 min at room temperature the excess of nucleotides were removed on Sephadex G-50 columns (Pharmacia) previously equilibrated with Tris/EDTA (1 mM EDTA, 25 mM Tris/HCl, pH 7.5). The concentration of the linear DNA was analyzed on an ethidium bromide stained 1 % agarose gel. After adjust- ment to 100 mM NaC1, approximately 100 ng of treated pRIT28 from the different reaction mixtures was subsequently incubated with streptavidin-coated paramagnetic beads (M-280, Dynal AS) for 15 min at room temperature followed by magnetic separation of the beads. The superna- tant from each sample was analyzed on an l % agarose gel, with approximately 100 ng HindIII-linearized pRIT28 used as reference. This procedure allowed a comparison of the polymerase activity of the different samples with respect to the dilution for which a decreased DNA-immobilization was observed.

RESULTS General concept

The basic principle for the competitive elution concept is outlined in Fig. 1. The target protein (X) is produced as a fusion to a suitable affinity tail (A). A sample (e.g. crude cell lysate) containing the recombinant fusion A-X is passed through a first column containing the ligand L, with affinity for the tail A. The recombinant fusion protein A-X is thus captured, enabling extensive washing to remove contaminants (Fig. la). A stoichiometric excess of a bifunctional competitor fusion protein A-B, where B is a second affinity tail capable of selective binding to a different ligand (LJ, is used for competitive elution of the A-X fusion at neutral pH (Fig. lb). Specific removal of competitor molecules from

Fig. 1. General concept for the competitive-elution strategy. See text for details. X, target protein; A, first affinity tail, used both fused to the product and in the competitor protein A-B; L,, ligand with affinity for A; B, second affinity tail used only in the competitor; L,, ligand with affinity for B.

the effluent mix is accomplished by a passage through a second affinity column containing the L, ligand, to which the A- B fusion is bound (Fig. lc). This concept thus allows specific affinity purification of sensitive recombinant gene products using very mild elution conditions.

Choice of fusion partners The synthetic IgG(Fc)-binding domain Z (Fig. 2a) de-

rived from staphylococcal protein A [33], has several fea- tures that make it suitable for use as an affinity-fusion partner. The domain shows high binding specificity and is relatively small (7 kDa), highly soluble, folding efficient, stable to proteolysis, secretion competent and does not contain any

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S s E D A B C

IgG binding I I

X M

Klenow

I I 'pi

77 kDa [pRIT50]

serum albumin binding

Staphylococcal protein A

Streptococcal protein G

B B 40 kDa [pRIT49] I IgG binding serum albumin binding

Fig. 2. Schematic representation of the two affinity tails and the fusion proteins Z-Klenow and ZZ-BB. (a) Origin of the two affinity tails used in the study. The Z domain is a 7-kDa synthetic analogue of one (domain B) of the five IgG-binding domains of staphylococcal protein A. The 25-kDa serum-albumin-binding region BB is derived from the repetitive A-B region of streptococcal protein G [34]. Also indicated is the extension of a small 5-kDa subfragment with serum-albumin-binding activity (see Discussion). (b) The Z-Klenow fusion containing the monovalent Z domain fused to the Klenow fragment (residues 324-928) of E. coli DNA polymerase 1, as encoded from the expression vector pRIT50. Shown below is the bifunctional competitor fusion protein ZZ-BB encoded by the expression vector pRIT49, containing a divalent IgG binding region (ZZ) fused to the serum-albumin-binding region (BB) of streptococcal protein G. Both fusion proteins were expressed intracellularly in E. coli under the control of the promoter and translation-initiation signals (eight amino acids, shaded boxes) from the E. coli trp operon.

Table 1. Binding data of Z and ZZ proteins for polyclonal hIgG. The values were derived from three independent runs.

Immobilized ligandlanalyte k,,,

Polyclonal hIgG Z zz

5.4 (to.7) x 104 2.7 (+0.3) x 2.0 (+0.1) x 107 1.1 (+o.i)x105 3.3 (+0.2) x 10-4 3.3 (+O.4)X1O8

cysteines [4, 51. Furthermore, a difference in binding strength for monoclonal IgG of monovalent and divalent fragments of Z has been described (Pharmacia Biosensor AB, Applica- tion note no. 303). However, since human polyclonal IgG is most often used as affinity resin for the purification of protein A fusion proteins [12], binding kinetics of monovalent and divalent Z to human polyclonal IgG were determined by real-time interaction analysis. Fragments Z and ZZ were expressed in E. coli and the binding kinetics to immobilized polyclonal hIgG was investigated (see Experimental Pro- cedures for details). The results show that the divalent ZZ protein binds with more than a tenfold higher overall affinity to the polyclonal hIgG, compared to the monovalent Z protein (Table 1). An overlay plot of sensorgrams (Fig. 3) regis- tered for the two proteins at the same molanty clearly dem- onstrates the slower dissociation from the surface for the ZZ protein, which indicates an avidity effect due to two, rather

than a single binding site for IgG. Taken together, the data suggest that for mild recovery of Z-containing proteins from either monoclonal or polyclonal IgG-affinity columns, a competitive elution strategy based on the difference in binding characteristics of the Z and ZZ proteins would be effective.

For the competitor protein, a second constituent with affinity for a second ligand is desired to allow specific affinity capture of the competitor after elution. The streptococcal surface receptor protein G is a bifunctional molecule with the capacity to bind to both IgG and serum albumins from different species [34, 351. The serum albumin region, designated BB (Fig. 2a), has been subcloned and used as affinity- fusion partner for the production of recombinant proteins [8, 281, which can be purified by HSA-affinity chromatography. To obtain a bifunctional competitor protein capable of effective displacement in combination with a second affinity func-

107

1 16500

; 16400 LT

16300 .

& 16200 . i7j E 16100.

m

f 15900 u)

c 20

1 2 3 4 5 6 7 8 9 M

f 94 + 67

f 43

+ 30

15800 0 100 200 300 400 500 600 700

Time (s)

Fig. 3. Sensorgrams from the biosensor analysis. Overlay plot of sensxgrams from the BIAcoreTM analysis showing the difference in dissociation kinetics of proteins Z and ZZ in the interaction with polyclonal hIgG. Following the injection of 25-pl pulses of Z and ZZ (1600 nM) over a 6000 RU polyclonal hIgG surface at a flow- rate of 5 pllmin, the dissociation phase in a flow of pure buffer was monitored. Note that the signal decrease (approximately 150 RU) immediately after the completed injection (at 350 s) corresponds to a difference in refractive index between sample solution and driving buffer. The sensorgrams were adjusted relative to each other in order to facilitate the comparison of the dissociation kinetics for the two proteins.

tion, the expression vector pRIT49 was assembled, encoding the construct ZZ-BB (Fig. 2b).

Production of fusion proteins

To evaluate the concept, the production and purification of a fusion between the Z domain and the Klenow fragment of E. coZi DNA polymerase I, was chosen as model system. The 1.8-kbp Klenow gene fragment (see Experimental Pro- cedures for details) was fused to the gene encoding a monovalent IgG-binding domain Z to yield the construct designated Z-Klenow (pRIT5O; Fig. 2b). The production and stability of the encoded Z-Klenow fusion protein was analyzed using a conventional IgG-affinity chromatography protocol, including elution at pH 3. Analysis by SDSPAGE of IgG-affinity-purified soluble proteins showed that the fusion (77 kDa) was stable (Fig. 4) and produced at approximately 50 mg/l (data not shown). The ZZ-BB protein was expressed in a soluble form intracellularly in E. coZi and purified in a one step procedure by HSA-affinity chromatography. Analy- sis by SDSPAGE of the purified soluble proteins showed that the competitor was almost exclusively of full-length (40 kDa; Fig. 4) and produced at approximately 40 mgA (data not shown).

Purification and competitive elution

A bacterial lysate containing approximately 13 mg of the Z-Klenow fusion protein product was applied to an IgG- Sepharose column for specific capture of the fusion protein. After washing to remove contaminants, a solution (pH 7.5) containing the fusion protein ZZ-BB was introduced for competitive elution of the Z-Klenow fusion. The applied amount of competitor corresponded to a fourfold molar excess of competitor to immobilized target protein, based on the result from a column saturation experiment (see Experi- mental Procedures). The eluted samples were collected at

f 14

Fig. 4. Results from the affinity purification of the Z-Klenow fusion protein using the competitive-elution strategy. Analysis by 12% SDSPAGE of proteins from different stages of the purification protocol. Lane 1, IgG-affinity purified (pH 3 eluted) Z-Klenow fusion protein; lane 2, HSA-affinity purified competitor protein ZZ- BB; lanes 3-6, proteins eluted from the IgG column by competition, corresponding to fractions 10, 15, 20 and 30; lane 7, proteins eluted from the IgG-column by pH 3; lane 8, proteins eluted from the HSA-affinity column; lane 9, proteins in the flow-through from the passage of the pooled fractions over the HSA-affinity column; M, marker proteins, with molecular masses in kDa indicated on the right.

regular intervals, and analyzed by SDSPAGE (Fig. 4). For the initially eluted samples, corresponding to one column volume, a small amount of the Z-Klenow fusion can be observed together with some minor proteins corresponding to degradation products of the competitor ZZ-BB (Fig. 4). As the elution proceeded, the ratio between the Z-Klenow fusion and the competitor increased to a maximum after two column volumes (Fig. 4), where the majority of the proteins were the target product. This indicates an effective displacement of the product obtained by the competitor protein ZZ-BB. As an increasing number of binding sites in the column are oc- cupied by the competitor protein, successive effluent fractions contain an increasing concentration of the ZZ-BB protein (Fig. 4). Subsequent elution of the column with pH 3, after completed competitive elution and washing, showed that the displacement was effective, since the eluted material almost exclusively contained the competitor protein ZZ-BB, with only small amounts of non-eluted Z-Klenow fusion (Fig. 4).

The fractions from the competitive elution were pooled and loaded directly onto an HSA-Sepharose column to capture the bifunctional competitor fusion from the effluent mix. Analysis of the proteins eluted by pH 3 from this second affinity column showed the expected band at 40 kDa, corresponding to the recovered competitor fusion protein ZZ-BB (Fig. 4). As expected this capturing step was effective, since no trace of the competitor can be seen in the flow-through, which contains the Z-Klenow fusion with high purity (Fig. 4). The Z-Klenow fusion protein obtained by the competitive elution was found to have the same specific activity as commercial Klenow fragment in a functional assay involv- ing filling in of protruding ends of Hind111 restricted plasmids (see Experimental Procedures). This is in contrast to Z- Klenow material eluted with low pH and immediately neutralized, which gives a purified product with a relatively low activity (data not shown).

DISCUSSION The results presented here show that the competitive elu-

tion strategy using the engineered bifunctional ZZ-BB fusion

108

can be applied to obtain a fully biologically active product of high purity by IgG-affinity chromatography, using physiological conditions at all steps in the procedure. The overall efficiency of the competitive elution strategy is influenced by several factors. A competitor protein containing two (or more) copies of the competitive affinity function is advanta- geous due to a pronounced avidity effect. A comparison of the overall affinity constants for IgG for the proteins ZZ and Z reveals a fivefold difference for each mole of binding sites. A consequence of this difference is probably reflected in the composition of the early fractions of the effluent, containing a high proportion of displaced Z-Klenow fusion protein compared to the competitor protein. To minimize the amount of competitor needed for competition, the column should pref- erably be almost saturated with the target fusion protein, resulting in an immediate displacement of target fusions by the competitor rather than an occupancy of free binding sites. The use of the serum albumin binding fragment, which binds strongly and with high specificity to HSA (Kaff= 1.4 X lo9 M-' ; Nygren, P.-A., unpublished results), ensures that the competitor can be effectively removed from the effluent mix. In addition, this competitor can be recovered by acid elution of the HSA column, lyophilized or neutralized and used repeatedly. The use of the stable ligands IgG and HSA for the affinity matrices makes this system cost effective, since the columns can be used for more than 100 cycles of binding and elution without loss of activity 1131. For other expression strategies based on affinity fusions, such as the commercially available FlagTM 1151, Strep-tag 11 61, glutathione-S-transferase 1181 or maltose-binding protein 1191 systems, mild and effective elution protocols based on the displacement from the affinity resin by a bifunctional competitor can also be envisioned. A small 46-amino-acid segment comprising the third A-repeat flanked by 13 and 9 residues from the flanking B2 and S regions, respectively (Fig. 2a) [35], has been shown to have HSA-binding activity with a K,, of 1.OX lo7 M-' (Nygren, P.-A., unpublished results), and is therefore, in combination with the divalent BB tail, suitable as a monovalent fusion partner in analogy with the Z/ZZ system.

In conclusion, the data presented here for a Z-Klenow fusion protein, shows that efficient elution of protein A fusion proteins from IgG columns can be achieved under physiological conditions by a competition strategy. The use of a bifunctional competitor protein, which can be specifically and effectively removed from the effluent mix by simple passage through a second affinity column, yields a pure and defined target product. This strategy can be used to avoid the problems concerning irreversible loss of structure and aggregation frequently encountered when using relatively harsh conditions needed to elute fusions purified by affinity capturing techniques.

We thank Drs Stefan S ta l , Peter Lind and Tomas Moks for valuable discussions and advice. This work was supported by grants from The Swedish National Board for Technical Development (NUTEK) .

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