1 Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm and 2 Department of Genetics, Stockholm University, S-106 91 Stockholm, Sweden
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Abstract |
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Keywords: circular dichroism/ion-exchange chromatography/molecular modelling/pI/protein A
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Introduction |
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Ion-exchange chromatography (IEC) is a widely used protein separation technique in both small laboratory-scale and large-scale purifications. Its widespread utilization depends on several factors: (i) IEC resins are robust and cheap compared with other chromatographic resins, (ii) the resin can withstand CIP conditions, including contact with 1 M NaOH for several hours, considered a gold standard for cleaning of columns on the industrial scale (Dasarathy, 1996; Sofer and Hagel, 1997
) and (iii) knowledge about scaling of ion-exchange experiments from small laboratory purifications to large-scale processes is extensive (Sofer and Hagel, 1997
).
The adsorption to an ion exchanger is dependent on the physical characteristics of the target protein, e.g. pI and the charge distribution. IEC has the potential to result in high-resolution separation of loaded molecular species, but the performance is dependent on the amount of contaminants with the same adsorption characteristics as the target protein. One way to improve the purification factor of a recombinant target protein is to change its charge distribution to allow for a stronger adsorption to the ion exchanger or adsorption under conditions unique to the target protein, thus allowing adsorption and washing under more stringent conditions. Egmond et al. (1994) showed that by increasing the number of charged amino acids on the surface of the protease SavinaseTM it was possible to affect the strength of adsorption to a cation exchanger. However, even small changes in a protein introduced for facilitated purification could lead to, e.g., an impaired function, lower production levels or increased immunogenicity if intended for therapeutic use.
A system that could be of more general use was first described by Sassenfeld and Brewer (1984), who used an `ion-exchange handle' consisting of six arginines fused to the C-terminal end of human urogastrone, allowing the fusion protein to be eluted at a higher NaCl concentration from a cation-exchange column than urogastrone itself. However, the fusion protein was relatively insoluble and the chromatographic purification was therefore carried out in the presence of 5 M urea. In addition, consecutive positively charged amino acids (arginine and lysine) have frequently been reported to be substrates for proteolytic degradation (Grodberg and Dunn, 1988; Hellebust et al., 1988
; Sugimura and Higashi, 1988
; Sedgwick, 1989
).
An important consideration in the design of ion-exchange handles is the use of basic or acidic amino acids, to allow for either cation- or anion-exchange chromatography. The preferred choice is largely dependent on the host utilized for protein production. One of most frequently used host organisms for recombinant protein production is Escherichia coli, owing to its simplicity and wide applicability. A translation of the open reading frames in E.coli K12 (Blattner et al., 1997) reveals that the proteome is distributed in a bimodal fashion along a pI axis (VanBogelen et al., 1997
), with one cluster of isoelectric points ranging from 4.5 to 7.5 and the other from 8.0 to 10.5. Taking the relative expression levels into account, more than 90% of the proteins have an isoelectric point between 4 and 7 (Link et al., 1997
). Therefore, ion-exchange handles with positive charge recruited from lysines or arginines would theoretically be ideal, as it should allow for cation-exchange chromatography at high pH values where there is potentially less adsorption of contaminating E.coli host cell proteins.
An alternative to the use of a charged peptide handle described by Sassenfeld and Brewer would be to utilize a protein domain capable of independent folding for accommodation of charge. It should have the potential to be more proteolytically stable while still being able to confer a strong charge polarity in a fusion protein. Such charged domains can either be designed de novo based on e.g. helical motifs (Hoshino et al., 1997; Szilak et al., 1997
) or by adding charges to an existing protein, recruited as a scaffold. In this work, we investigated the three-helix bundle domain Z as a scaffold for the accommodation of positive charge to allow for cation-exchange chromatography at high pH values.
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Materials and methods |
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Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on an HP 1090 instrument from Hewlett-Packard (Waldbronn, Germany) using a C-18 column. All ion-exchange experiments were performed on an Äkta-Explorer 100 system (Amersham Pharmacia Biotech).
Molecular modelling
Molecular modelling was performed on a Silicon Graphics Octane workstation (Access Graphics, Amstelveen, The Netherlands) using Sybyl (Tripos, St. Louis, MO). Energy minimization was carried out using a Kollman-all force field (Weiner et al., 1984, 1986
) with standard parameters. The model of Zwt was constructed by adding hydrogens to the N-terminus of the NMR structure (Protein Databank entry 2SPZ, http://www.rcsb.org/pdb). Charges were loaded on to the molecule and five layers of water were added to the system. Energy minimization was carried out while keeping Zwt static, followed by energy minimization of the whole system. The mutants were constructed by exchanging the appropriate amino acids, followed by their manual alignment. Charges were loaded on to the system and it was energy minimized while keeping the backbone and non-mutated residues static. Five layers of water were added and energy minimization was performed while keeping the protein static and finally the whole system was energy minimized. Electrostatic surface potentials were calculated using Delphi (Gilson and Honig, 1987
; Nicholls and Honig, 1991
). Molecular surfaces were built in Grasp (Nicholls et al., 1991
).
Assembly of Z variants
Methods for recombinant DNA work were performed essentially as described by Sambrook et al. (1989). Oligonucleotides coding for helices 1 and 2 of the three Z variants were assembled in a stepwise fashion analogous to the Z-library constructed by Nord et al. (1995). The base compositions of the constructs were verified and the resulting vectors were labelled pKN1Zwt, pKN1Zbasic1 and pKN1Zbasic2, respectively. Each one codes for one Z variant followed by a streptococcal Protein G-derived albumin-binding domain (ABD) (Eliasson et al., 1991).
Construction of expression vectors
The vectors pKN1Zwt, pKN1Zbasic1 and pKN1Zbasic2 was used as templates in PCR {[96°C for 15 s; 59°C for 20 s; 72°C for 90 s (30 times)], 72°C for 7 min} using the primers Zsub1: 5'-CCCCGAATTCCGTAGACAACAAATTCAACAA-3' and GRTO-10: 5'-CCGGCCGGCTGCAGTTAATGGTGATGGTGATGGTGTTTCGGCGCCTGAGCATCATTTAG-3'. The resulting PCR product was ligated to pGEM-5zf(+) (Promega, Madison, WI) and base composition was verified. The pGEM-5zf(+) construct was then restricted with EcoRI and PstI and the genes coding for the Z variants were isolated and ligated with pTrpABD (Kraulis et al., 1996) that had been cut with the same enzymes. The resulting vectors each code for a Z variant including a trp-leader followed by a His6-tag under control of the trp-promoter. The different vectors were named pTrpZwt, pTrpZbasic1 and pTrpZbasic2.
Production and purification of Zwt, Zbasic1 and Zbasic2
The pTrp constructs were grown at 30°C for 20 h in 1 l of TSB + YE medium (30 g/l tryptic soy broth (Lab M, Topley House), 5 g/l yeast extract (Difco), 50 mg/l kanamycin). Zwt was purified using IgG affinity chromatography as described (Nilsson et al., 1987), followed by RP-HPLC. Cell cultures containing Zbasic1 or Zbasic2 were centrifuged and the resulting pellet was resuspended in ~30 ml of buffer A (10 mM phosphate, pH 7) supplemented with 450 mM NaCl. The cells were then disrupted by sonication followed by centrifugation to remove cell debris. The supernatant containing soluble intracellular material was filtered through a 1.2 µm hydrophilic filter (Sartorius, Göttingen, Germany) and loaded on a 20 ml Q-Sepharose FF column, equilibrated with buffer A supplemented with 450 mM NaCl. The flow-through (50 ml) was collected and diluted to 300 ml with buffer A and loaded on a 20 ml S-Sepharose FF column that had been equilibrated with buffer A. The column was washed extensively and bound proteins were eluted using a linear NaCl gradient from 0 to 1 M. Fractions containing Zbasic1 or Zbasic2 were pooled and further purified by RP-HPLC. The purity of Zwt, Zbasic1 and Zbasic2 was determined by loading ~4 µg of each protein on a 16% TrisTricine gel (Novex Electrophoresis, Frankfurt, Germany) and running it according to the manufacturer's recommendations (see Figure 3
).
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Data were collected using a Jasco J-720 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a thermostable water-bath for temperature control. The buffer used in all experiments was 10 mM phosphate, pH 7.0. Scan data were collected between 250 and 190 nm at 25°C with a scanning speed of 50 nm/min and each spectrum is an average of three consecutive scans. Data for the thermal melts were collected as the change in 222 nm. The temperature gradient was 60°C/h.
Cation-exchange analysis of Zwt, Zbasic1 and Zbasic2
Cation-exchange analysis was performed on the purified proteins, by loading ~1 mg of each protein on to a 1 ml Resource S column (Amersham Pharmacia Biotech) that had been equilibrated with 5 column volumes (CV) of running buffer (50 mM phosphate at pH 3 and 20 mM ethanolamine at pH 9). The column was washed with 1015 CV of running buffer, with subsequent elution with a linear gradient of 02 M NaCl for experiments performed at pH 3 or 01 M NaCl for all other experiments. The length of the NaCl gradient was 20 CV in all experiments and the chromatograms were recorded as the absorbance at 214 nm.
Production and purification of Zbasic2ABD
Cells containing pKN1Zbasic2 were grown in 1 l batches in TSB + YE medium supplemented with ampicillin (100 mg/l) at 30°C for 24 h with induction by adding 240 mg of isopropyl-ß-D-thiogalactoside (IPTG) at OD = 1. Cells were harvested, lysed by sonication, followed by affinity chromatography on human serum albumin (HSA) Sepharose as described by Nygren et al. (1988). Eluted fractions were pooled, freeze-dried and redissolved in 20% acetonitrile in water supplemented with 0.25% (v/v) of pentafluoropropionic acid. Zbasic2ABD was further purified using RP-HPLC.
Cation-exchange chromatographic analysis of Zbasic2ABD
Purified Zbasic2ABD fusion protein was loaded on a 1 ml Resource S column previously equilibrated to the different pH values investigated. The column was washed with 10 CV of equilibration buffer and elution was performed using a linear gradient of 01 M NaCl. The buffers used were 20 mM 1,3-diaminopropane, pH 8.5 and 10.5, 20 mM ethanolamine, pH 9.5, 50 mM phosphate, pH 7.5, and 20 mM bis-Tris propane, pH 6.5.
Purification of Zbasic2ABD from a whole cell lysate
Purified Zbasic2ABD fusion protein was spiked into a whole cell lysate from E.coli (50 µg fusion protein/ml cell lysate). The mixture was loaded on a 1 ml Resource S cation-exchange column equilibrated with running buffer (50 mM phosphate, pH 7.5). The column was washed with running buffer and elution was performed using a linear gradient of NaCl from 0 to 1 M.
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Results |
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In this work we used the Z domain as a scaffold for creating highly charged protein domains. The Z domain is a compact 58 amino acid (7 kDa) three-helix bundle derived from the B domain of staphylococcal protein A (Nilsson et al., 1987). It contains no cysteines and is highly soluble. The Z domain has been shown to help in solubilizing fused target proteins in vitro (Samuelsson et al., 1994
) and is also stable against proteolysis in a number of different hosts (Ståhl and Nygren, 1997
). In addition, this domain has earlier been shown to be highly permissive to mutations in helices one and two in studies where it was used as a scaffold for combinatorial mutagenesis to isolate variants with novel binding specificities using phage display selection technology (Nord et al., 1995
, 1997
). An indication that the Z domain could also accommodate numerous basic amino acids on its surface came from panning experiments against the highly acidic target protein EB200, derived from P.falciparum (Ahlborg et al., 1997
), in which a highly basic variant (ZEB4) containing multiple positively charged amino acids was selected (Figure 1
). That variant was shown to have a secondary structure content similar to that of the Zwt protein as determined by circular dichroism spectroscopy (E.Gunneriusson, personal communication). However, ZEB4 contained two cysteines at positions 25 and 32 that were undesirable in a purification handle. Therefore, the first gene construct encoding a highly charged ion-exchange handle was ZEB4, with the two point-mutations C25S and C32S, designated Zbasic1. Its amino acid sequence is shown in Figure 1
.
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Molecular modelling of Z variants
Based on the solution structure of Z (Tashiro et al., 1997), energy-minimized models of Zwt, Zbasic1 and Zbasic2 were calculated (data not shown). The model of Zwt shows a compact anti-parallel three-helix bundle very similar to the starting NMR structure. The models of Zbasic1 and Zbasic2 have similar backbone traces but Zbasic2 has a larger molecular surface owing to its many arginines. Zbasic1, on the other hand, has a smaller molecular surface than Zwt. On close examination of the structures, we note that the arginines can participate in multiple hydrogen bonds, both with the backbone and with other amino acids in their vicinity, that was not possible for their substituents in Zwt. Electrostatic potentials for the molecular surfaces of the three Z variants were also determined and are displayed in Figure 2
. This shows Zwt to have a slightly hydrophobic molecular surface with about equal elements of positive and negative potential. Zbasic1 has a less hydrophobic molecular surface and a large positively charged area with islands of negative charge interspersed. Zbasic2 has an area (of ~500 Å2) with extremely high positive charge.
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Zwt, Zbasic1 and Zbasic2 were successfully expressed as intracellular proteins in E.coli. Zwt was purified on a resin containing immobilized IgG for capture and purification (Nilsson et al., 1987), after which it was ~95% pure. Zbasic1 and Zbasic2, on the other hand, do not bind IgG since several of the amino acids involved in IgG binding (Deisenhofer, 1981
; Jendeberg et al., 1995
) has been changed. They were instead purified using cation-exchange chromatography at pH 7 and purified to 90% purity for Zbasic1 and >95% purity for Zbasic2 (data not shown). The material from the initial purification of the three variants was further purified by RP-HPLC to more than 95% purity, as can be seen in Figure 3
.
Structural characterization of Zwt, Zbasic1 and Zbasic2
In order to characterize the secondary structure of the Zbasic variants, their absorption of far-ultraviolet circular polarized light was measured between 190 and 250 nm. The CD spectra collected for Zwt, Zbasic1 and Zbasic2 are shown in Figure 4. The double minima for all three proteins at 222 and 209 nm and a high peak around 195 nm are characteristic for proteins with a high
-helical content. Tashiro and co-workers have determined the structure of Zwt and they have assigned 65% of the amino acids in
-helical conformation (Tashiro et al., 1997
). The
222 nm response for the mutants are ~70 and ~80% for Zbasic1 and Zbasic2, respectively, of the response for Zwt. Based on the similarity of the three spectra, we concluded that both Zbasic1 and Zbasic2 also have a high
-helical content although lower than that of Zwt, which is to be expected owing to the numerous mutations introduced.
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The thermal denaturation of the three Z variants is shown in Figure 5. The melting curves show only one transition region each. Therefore, a two-state folding mechanism was assumed. The melting points were calculated using standard techniques (Creighton, 1997
) and are displayed in Table I
. Zwt melts at the highest temperature (75°C) while Zbasic1 melts at a lower temperature (62°C) and Zbasic2 melts at the lowest temperature (40°C). All three proteins show a slow loss of
-helicity at the pre- and post-transition regions.
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Purified proteins (1 mg) were loaded on a cation-exchange column. At pH 3 all three proteins were binding quantitatively to the resin as shown in Figure 6A using linear NaCl gradient elution. Zwt was eluted at an NaCl concentration corresponding to a conductivity of 60 mS/cm, whereas Zbasic1 was more tightly bound to the ion-exchanging groups and therefore eluted at a higher NaCl concentration corresponding to 80 mS/cm. Zbasic2, the most basic variant, was eluted at 110 mS/cm corresponding to the highest NaCl concentration. At pH 9 the Zwt protein was only detected in the flow-through (Figure 6B
), whereas both Zbasic1 and Zbasic2 were still quantitatively adsorbed at this pH. Both mutants were eluted at lower NaCl concentrations than at pH 3, corresponding to 20 and 40 mS/cm, respectively (Figure 6B
). These ion-exchange results showed that Zwt was positively charged at pH 3 but negatively charged at pH 9 (Figure 6A and B
). Zbasic1 and Zbasic2, on the other hand, were positively charged at both pH 3 and 9 (Figure 6A and B
). Zbasic1 lost its binding to the cation-exchange column when the pH was increased above 9. Zbasic2 was completely adsorbed on the column at both pH 10 and 11. However, at pH 11 the amount of Zbasic2 eluted from the column was significantly reduced. The pI values were also measured using isoelectric focusing and the values are shown along with theoretically calculated values in Table I
. The isoelectric point for Zwt was determined to 6.4 and for Zbasic1 to 8.2. Zbasic2 focuses at the negative electrode since its pI is higher than that which can be resolved on the gel used (pH 3.5 to 10). Its pI was determined as >9.3 as that corresponds to the marker of highest pI that can be resolved on the gel.
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In order to investigate if the chromatographic properties of the Zbasic2 domain were preserved also after fusion to a target protein, Zbasic2 and an albumin-binding domain (ABDpI 5) derived from streptococcal protein G were fused on the genetic level. The gene fusion, Zbasic2ABD, was expressed as a periplasmically secreted protein and the resulting gene product was purified to homogeneity (see Materials and methods). Purified Zbasic2ABD fusion protein was loaded on a cation-exchange column at different pH values and eluted with a linear gradient of NaCl from 0 to 1 M. As previously seen for the free Zbasic2 domain, this fusion protein could be recovered by cation exchange at pH values ranging from 6.5 to 10.5 (see the example in Figure 7). However, at pH > 7.5, apparently lower yields were obtained (see Discussion).
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In order to investigate the usefulness of the positively charged Zbasic2 domain as a purification tag, Zbasic2ABD fusion protein was spiked into a total cell extract from E.coli and loaded on a cation-exchange column at pH 7.5. The resulting chromatogram (Figure 8) demonstrates that it was possible to capture the protein by the cation-exchange column and also that the selectivity was high, leading to a product of >90% purity.
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Discussion |
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Both new variants could be produced in E.coli and purified by different means, indicating that they were stable molecules and correctly folded despite their high content of positively charged amino acids. Far-ultraviolet CD measurements on the mutants showed that their -helical content is of the same order of magnitude as that of their parental molecule Zwt. Thermal denaturation of the mutants showed that they have significantly lower melting points than the wild-type protein. This indicates that it is favourable to start with a scaffold with high thermal stability, as was done in this work to ensure that the stability of the protein after charge engineering does not decrease to very low values.
An interesting observation was the highly varied migration in the SDS electrophoresis analysis of the various variants (Figure 3). The theoretical molecular weights differ by only 0.5 kDa between the variants, while the apparent sizes, judged by the electrophoresis, differ by ~2 kDa. Analysis using electrospray mass spectrometry confirmed that the proteins have molecular weights identical with the theoretical values.
A comparison between the wild-type Z domain and the engineered variants in cation-exchange experiments demonstrated a dramatic difference in chromatographic performance. At low pH values the basic variants were eluted at much higher salt concentrations than the wild-type variant and at elevated pH values only the engineered variants could be adsorbed on the chromatographic resin (Table II). These pH-dependent binding properties followed the expected differences in surface charge properties (Figure 2
). Interestingly, the Zbasic2 variant retained its binding properties also when fused to the acidic fusion partner ABD. At pH values up to 7.5 the Zbasic2ABD fusion protein was completely adsorbed and quantitatively eluted from the cation-exchange resin. When experiments were performed at pH > 7.5, smaller amounts of fusion protein were eluted from the column. This behaviour could possibly be explained by the documented instability to alkaline conditions shown for the ABD domain (Gülich et al., 2000
). Nevertheless, these results indicate that the Zbasic2 domain is capable of serving as a functionally independent domain in the fusion protein. This notion is further supported by the fact that it was initially possible to purify the Zbasic2ABD fusion protein using HSA-Sepharose chromatography (see Materials and methods), showing that the fused ABD domain folded correctly and was not influenced by its fusion partner Zbasic2 to any great extent.
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These results show upon the power of protein design to create protein domains with desired properties starting from suitable scaffold structures. The properties of the basic variants used here suggest that they could be interesting to investigate for broad use as fusion partners for cation-exchange chromatographic purification.
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Notes |
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Acknowledgments |
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References |
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Received January 7, 2000; revised July 26, 2000; accepted August 10, 2000.