Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden
1 To whom correspondence should be addressed. e-mail: sophia{at}biotech.kth.se
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Abstract |
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Keywords: affinity chromatography/capacity/protein G/purification/serum albumin-binding domain
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Introduction |
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We have previously described the use of an albumin-binding domain (ABD) derived from streptococcal protein G for efficient capture of HSA using affinity chromatography. Protein engineering was used to replace alkali-sensitive residues in ABD to create a new ligand, denoted ABD*, that meets industrial requirements for column cleaning-in-place (CIP) procedures based on alkaline exposure (Asplund et al., 2000) and therefore has potential for use in large-scale HSA purification (Gülich et al., 2000
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The aim of this study was to develop second-generation ligands based on the ABD* protein. Using genetic engineering, head-to-tail dimeric versions have been constructed and further equipped with a unique C-terminal cysteine residue providing a tool for directed coupling of ligands to chromatographic resins. The effect on binding capacity from the dimerization and alternative coupling chemistries has been investigated by both biosensor and affinity chromatography techniques. In addition, the influence on the alkaline stability through the use of three different connective linker sequence candidates has been evaluated.
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Materials and methods |
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For dimerization of ABD*, the plasmid pTrpABD* (Gülich et al., 2000) was used as template in a standard polymerase chain reaction (PCR) protocol. The same vector was used for expression of the monovalent ABD*. Oligonucleotides were purchased from Interactiva (Ulm, Germany) and the cloning was performed using standard protocols according to Sambrook et al. (Sambrook et al., 1989
). Before dimerization of ABD*, a PCR step to introduce a C-terminal cysteine to allow directed immobilization using the free C-terminal thiol was carried out. The PCR fragment was inserted into plasmid pTrpABD* using the XbaI and PstI restriction sites, to generate pTrpABD*cys. The non-palindromic restriction enzyme AccI was used to steer head-to-tail dimerization of ABD. For the first divalent constructs, ABD*dimerA and ABD*dimerB, the ABD fragments were equipped with AccI sites at both the 3'- and 5'-ends in the PCR step. For the construction of the ABD*dimerB, the asparagine in the pre-existing linker sequence VDANS present at the N-terminus of ABD* was substituted for an aspartatic acid in the PCR step to yield the VDADS linker. This PCR product was ligated into the pGEM-T vector system I (Promega, Madison, WI) and sequenced.
The pGEM-T plasmid carrying the correct ABD* fragment was restricted and ligated in a head-to-tail fashion into plasmids pTrpABD*A and pTrpABD*B predigested with AccI, giving rise to pTrpABD*dimA and pTrpABD*dimB, respectively. Plasmid pTrpABD*dimC was constructed by using two-step PCR (Higuchi et al., 1988) with pTrpABD*dimA as template. The gene fragment encoding ABDdimerC was purified and introduced into pTrpABD* cleaved with XbaI and PstI. A MegaBACE 1000 DNA Sequencing System (Amersham Biosciences, Uppsala, Sweden) was used to verify the correct sequence of the inserted fragments. MegaBACE terminator chemistry (Amersham Biosciences) was utilized according to the suppliers recommendations in a cycle sequencing protocol based on the dideoxy method (Sanger et al., 1977
). The ligation mixtures were transformed to Escherichia coli strain RR1
M15 (American Type Culture Collection, Rockville, MD), which was also used for expression of the gene products.
Production and purification
E.coli cells harboring the different ABD* variants were used to inoculate 20 ml of tryptic soy broth (30 g/l) (Difco, Detroit, MI) supplemented with 5 g/l yeast extract (Difco) and 50 mg/l kanamycin monosulfate (LabKemi, Stockholm, Sweden) and were incubated overnight at 37°C. A 5 ml volume of the overnight culture was used to inoculate 500 ml of fresh medium. The culture was incubated overnight at 37°C and the cells were harvested by centrifugation (5000 g for 10 min). After resuspension in TST buffer (25 mM TRISHCl, pH 7.5, 150 mM NaCl, 1.25 mM EDTA, 0.05% Tween 20), the cells were disintegrated by sonication (Sonics and Materials, Danbury, CT). For clarification a centrifugation step was carried out (30 000 g for 20 min) and the lysate was filtered (0.45 µm) (Millipore, Bedford, MA).
Proteins were purified using HSA affinity chromatography on an ÄKTA Explorer 10 (Amersham Biosciences) purification system. After washing with TST buffer and 5 mM NH4OAc, pH 5.5, the bound product was eluted with 0.5 M HOAc, pH 2.8. The protein was detected by absorbance measurements at 280 nm and relevant fractions were lyophilized.
The homogeneity was analyzed by SDSPAGE on high-density gels (Amersham Biosciences) using the Phast system (Amersham Biosciences) under reducing conditions and stained with Coomassie Brilliant Blue (Amersham Biosciences).
Biospecific interaction analysis
A BIAcore 2000 instrument (BIAcore, Uppsala, Sweden) was used for real-time biospecific interaction analysis. Lyophilized protein samples were dissolved in HBS buffer (10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4) and filtered (0.45 µm) (Millipore). ABD* and ABD*dimerA were immobilized by N-hydroxysuccinimide and N-ethyl-N'-(3-diethylaminopropyl)carbodiimide chemistry to the carboxylated dextran layer of a CM5 sensor chip (BIAcore) according to the suppliers recommendations. The immobilization resulted in 900 RU for each variant. Duplicate samples of 300 nM HSA (Pharmacia, Stockholm, Sweden) in HBS were injected over the surfaces at a flow rate of 10 µl/min.
Prior to immobilization, the cystein-containing ligands were reduced in 10 mM dithiothreitol (DTT) in TST buffer, followed by buffer exchange on Sephadex G-25 columns (Amersham Biosciences) to 50 mM NH4OAc, pH 4.0. The free thiol of the C-terminal cysteine was used for directed coupling of ABD* and ABD*dimerA to the sensor chip. 2-(2-Pyridinyldithio)ethanamine hydrochloride (PDEA) (BIAcore) was injected over an NHS/EDC activated CM5 sensor chip (BIAcore). The immobilization resulted in 900 RU for ABD* and ABD*dimerA. Duplicate samples of 300 nM HSA (Pharmacia) in HBS were injected at a flow rate of 10 µl/min.
For all analyses, 10 mM HCl was used to regenerate the surfaces. The data were analyzed using the BIA evaluation 3.0.2b software (BIAcore). The signals from a non-immobilized surface (activated and deactivated) were subtracted.
NaOH treatment of the different constructs analyzed by SDSPAGE analysis
Lyophilized samples were dissolved in 0.5 M NaOH. After incubation for 180 min at room temperature the proteins were precipitated with acetone. The proteins were then dissolved in 1*RED (20 mM TRISHCl, pH 8.0, 1 mM EDTA, 2.5% SDS, 5% ß-mercaptoethanol, 0.01% BFB) and analyzed by SDSPAGE according to manufacturers recommendations (Amersham Biosciences).
Affinity chromatography with CIP treatment
ABD* and the different ABD*dimer variants were covalently coupled to an agarose matrix using thioether chemistry (Amersham Biosciences). The different gels were packed in HR5 columns (Amersham Biosciences) for analysis of the stability towards CIP treatment. An ordinary affinity purification protocol was followed on an ÄKTA Explorer 10 (Amersham Biosciences) instrument. HSA (10 mg/ml) (Pharmacia) in TST was loaded at a flow rate of 1 ml/min. After extensive washing with TST, the protein was eluted with 0.5 M HOAc, pH 2.8, supplemented with 100 mM NaCl. NaCl was included to disrupt proteinprotein interactions and thereby obtain a narrower peak. After elution the columns were washed with 0.1 M NaOH for 20 min. This procedure was repeated for 21 cycles to follow the decrease in capacity of the columns. The capacities of the columns were determined by measuring the absorbance of the eluted peak. The amount of eluted HSA was also determined by amino acid analysis (BMC, Uppsala, Sweden).
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Results |
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Biospecific interaction analyses
Biosensor binding studies were performed to elucidate any differences in binding capacity between the monovalent original construct ABD* and the divalent variant, ABD*dimerA. In addition, both a directed (thiol coupling) and a non-directed approach (amine coupling) were investigated in parallel to evaluate the importance of the chemistry used for coupling of the ligand to the sensor chip. Approximately equal amounts [measured in resonance units (RU)] of the different ligands were immobilized, thus corresponding to equimolar amounts of ABD* moieties (i.e. number of domains), presented as either monovalent or divalent constructs. The sensorgrams recorded during injection of HSA as analyte over surfaces carrying the respective ligands coupled by non-directed amine coupling chemistry reveal that a divalent ABD* ligand displays a higher dynamic binding capacity than its monovalent counterpart (Figure 2A).
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Column affinity chromatography studies
The different divalent ABD* variants, and also the monovalent ABD* domain, were separately coupled to activated Sepharose via their C-terminal cysteines to form directed thioether linkages. The resulting ligand densities of the chromatography matrices were ABD* 3.0 mg/ml, ABD*dimerA 3.4 mg/ml, ABD*dimerB 5.1 mg/ml and ABD*dimerC 2.6 mg/ml (Table I). These affinity media were subsequently used for HSA-binding capacity studies in a standard affinity chromatography protocol involving washing with TST buffer and elution at low pH. The results showed that columns containing divalent ABD* ligands had similar dynamic binding capacities for HSA as calculated per domain, compared with the column containing the monovalent ABD* ligand (Table I). These results corroborated the biosensor studies and indicated that both ABD* moieties in the divalent constructs were equally accessible for interaction with HSA.
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The divalent ligand constructs with different connective linker sequences were investigated for their stability towards alkaline sanitization procedures by incubation of the purified proteins in 0.5 M NaOH (pH 13.7) for 3 h, followed by analysis by SDSPAGE. Figure 3 shows that incubation of the divalent ligands at pH 13 resulted in the appearance of varying amounts of lower molecular weight bands, presumed to correspond to degradation products of full-length ligands. For variant C (Figure 3, lane 6), a relatively strong band which co-migrates with the monovalent ABD* reference protein is observed (Figure 3, lane 7). This suggests that the GGGSG linker sequence in this variant was relatively unstable to the treatment and that degradation of some of the material into monovalent ABD* domains occurred. Alkaline treatment of variant A also resulted in the appearance of some degradation products, but with a more even size distribution (Figure 3, lane 4). Interestingly, variant B showed the least degree of degradation compared with the other two variants, suggesting that this variant was the most stable to the alkaline treatment.
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To investigate if the same performance that the ligand variants demonstrated in 0.5 M NaOH was also observed when coupled to the chromatographic resin, a series of multiple-cycle affinity chromatographic experiments under repeated exposure to 0.1 M NaOH were performed.
Each chromatographic cycle consisted of the application of excess HSA to the columns prior to washing, followed by the measurement of the amount of eluted material to determine any decrease in binding capacity of the different resins. A 20 min pulse of 0.1 M NaOH was applied to the columns between each cycle to achieve a total ligand exposure time of 7 h. Interestingly, the results, summarized in Figure 4, showed that the alkaline stability of the ligands ABD*, ABD*dimerA and ABD*dimerC did not vary significantly, suggesting that the two linker variants in these divalent constructs were not affected to a greater extent than the flanking ABD* domains under these conditions. In fact, these ligands retained as much as 85% of the original capacity after 7 h of exposure to 0.1 M NaOH, Interestingly, the ABD*dimerB showed a stability towards high pH exceeding even the monomeric variant and retained as much as 95% of the capacity after 7 h of exposure to 0.1 M NaOH (see Discussion).
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Discussion |
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The three divalent constructions with different linker sequences (VDANS, VDADS, GGGSG) were evaluated for use in affinity chromatography. Both alkaline incubation and column chromatographic/alkaline sanitization experiments indicated that all three ligands possess a remarkably overall high stability, but that the ligand containing the VDADS linker was the most stable under high-pH conditions. Surprisingly, the column chromatographic experiment showed that the divalent ligand containing this linker sequence was even more stable than the monovalent ABD* ligand. The reason for this remains unclear, but it cannot be ruled out that the two domains in a divalent ligand can excert a mutual stabilizing effect related to refolding after exposure to denaturing conditions (Robinson and Sauer, 1998; Wenk et al., 1998
). Hence it is not only the linker length and composition but also the function of the separate domains within the protein that are essential for the stability of the ligand. A recently published analysis of different naturally occurring inter-domain linkers shows that the most frequently occurring length is 610 amino acids. However, both length and structure differ owing to the properties of the connected domains (George and Heringa, 2003
). Hence the choice of linker region, i.e. length and composition, is important when connecting two functional domains. Moreover, the choice of coupling chemistry is also of great importance in order to retain the functionality of the connected domains. Our data show that the use of directed coupling results in twice as many active domains for the divalent constructs compared with non-directed coupling. Notably, the difference between directed and undirected coupling is even greater for the monovalent versions of the linker domains.
These results clearly demonstrate the importance of the choice of coupling chemistry and linker sequence to achieve an effective presentation of the functional domain to its target molecule. In addition, they also demonstrate that the designed ABD*dimer with the VDADS linker is highly tolerant towards alkaline treatment and could therefore be a very suitable ligand candidate for the large-scale purification of HSA.
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Acknowledgements |
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References |
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Received May 12, 2003; revised October 8, 2003; accepted October 21, 2003