A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products

Journal of Biotechnology 99 (2002) 41 /50 www.elsevier.com/locate/jbiotec

A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products Susanne Gra¨slund, Malin Eklund, Ronny Falk, Mathias Uhle´n, ˚ ke Nygren, Stefan Sta˚hl * Per-A Division of Molecular Biotechnology, Department of Biotechnology, Royal Institute of Technology (KTH), SCFAB, SE-10691 Stockholm, Sweden Received 2 January 2002; received in revised form 8 May 2002; accepted 17 May 2002

Abstract An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein ASepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Affibody; Affinity blotting; Affinity chromatography; cDNA; Combinatorial protein engineering; Gene expression; Proteomics; Staphylococcus aureus protein A

1. Introduction

* Corresponding author. Tel.:
/46-8-5537-8329; fax:
/46-85537-8481 E-mail address: [email protected] (S. Sta˚hl).

The efforts to characterize proteins from entire proteomes that are presently discussed (Anderson et al., 2001), clearly demonstrate the importance of high-throughput protein expression systems. Ideally, such systems should allow for the production

0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 1 5 8 - X

42

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

and purification of a large number of proteins with different properties and not be optimized for a single protein (Larsson et al., 2000). In addition, the possibility to streamline the procedure from cloning to purification to enable automation will most probably be required for proteome-wide expression efforts. A large number of affinity gene fusion tag systems are available, that have been employed to create expression systems allowing for a common purification strategy irregardless of the properties of the target protein (Uhle´n et al., 1992; Nilsson et al., 1997). These systems are based on different types of interactions including, protein/ protein interactions, enzyme-substrates, antibody /antigens, carbohydrate-binding proteins/ carbohydrates, polyamino acids /ion exchangers or metal chelating ligands (Uhle´n et al., 1992). The hexahistidyl (His6) tag which can be recovered using immobilized metal-ion affinity chromatography, IMAC (Porath et al., 1975), is one of the most widely used systems, due to its simplicity and ability to be used under denaturing conditions. However, a drawback of IMAC is that it is based on a non-biospecific interaction which makes it less stringent than methods based on proteinprotein interactions (Uhle´n et al., 1992). Today, one of the most common affinity gene fusion system, using a protein affinity tag, is the Glutathione S-Transferase (GST) system (Simons and Vander Jagt, 1981; Smith and Johnson, 1988). Another widely used affinity tag is the FLAG peptide which employs monoclonal antibodybased affinity resins (Hopp et al., 1988). A welldocumented system is based on the interaction between protein A, or derivatives thereof, and the Fc fragment of immobilized IgG ligands (Uhle´n et al., 1983; Sta˚hl et al., 1999a). This system has proven effective for the production and purification of recombinant fusion protein in a variety of host cells including bacteria, yeast, insect cells, plant cells and mammalian cells (Sta˚hl et al., 1999a). The compatibility of protein A-based fusion partners with product secretion, the absence of disulfides and the inherent high solubility and proteolytic stability of such fusion partners, are important factors in bioprocess development considerations (Sta˚hl et al., 1999a; Nilsson et al., 1997;

Hansson et al., 1994). A drawback of the protein A-IgG affinity fusion system is that the immobilized IgG ligand used is relatively fragile, frequently associated with leakage of antibody subdomains (H and L chains) into the product eluate. In addition, commercially available IgG resins consist of polyclonal immunoglobulin preparations derived from human donors, which calls for viral contamination considerations if products are intended for therapeutic use. Although recombinant sources of protein A-binding Fc fragments can be envisioned (Jendeberg et al., 1996), the disulfide-bridged two chain structure of such subfragments is still a limitation when column sanitation protocols involving high pH are considered. Advances in peptide and protein library technology has made it possible to select new ligands with novel binding specificities and high endurance to chemical and physical conditions. One described strategy is the use of a Staphylococcus aureus protein A (SPA)-derived three-helix bundle domain as scaffold for protein library constructions. Through random mutagenesis of 13 surfacelocated residues of this 58-residue three-helix bundle, libraries have been constructed from which novel affinity proteins towards a wide range of protein targets have been selected using phage display technology (Nord et al., 1995, 1997, 2000; Gunneriusson et al., 1999a,b; Hansson et al., 1999). Obtained binding proteins, denoted Zaffibodies, show high binding specificities for their respective targets and have proven useful for example as robust ligands in affinity chromatography purification settings, for one-step recovery of target proteins from crude preparations including bacterial total cell lysates or CHO cell culture derived feed streams (Nord et al., 2000, 2001). Here, we present a novel affinity gene fusion system allowing purification on protein A-Sepharose, a robust and well-characterized chromatographic media, taking advantage of an affinity tag ZSPA-1 developed by combinatorial protein engineering (Eklund et al., 2002). To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were evaluated for convenient screening of expression efficiencies. Expression

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

levels and purification efficiency have been investigated. Furthermore, the capability of the ZSPA-1 / SPA interaction to withstand partly denaturing conditions for recovery of proteins with low solubility was investigated. The general use of the presented expression system in high-throughput protein expression efforts is discussed.

2. Materials and methods 2.1. Bacterial strains and DNA preparation DNA constructions were performed in Escherichia coli strain RRIDM15 (Ru¨ther, 1982) and expression of fusion proteins was performed in E. coli strain BL21(DE3)pLysS (Novagen Inc., Madison, WI). All oligonucleotides were manufactured by Interactiva Biotechnology GmbH (Ulm, Germany). Plasmid preparations were performed with Jet Star-columns (Genomed, Inc., Research Triangle Park, NC) and purification of gene fragments was done with GenEluteTM Agarose Spin Columns (Sigma, Inc., St. Louis, MO). All enzymes used were manufactured by MBI Fermentas AB (Vilnius, Lithuania), except Asc I, that was manufactured by New England Biolabs Inc. (Beverly, MA). 2.2. Construction of vector pAff11c The vector pAff8c (Larsson et al., 2000) was restricted with Kpn I and Nhe I and dephosphorylated. A gene fragment from the vector pT7-ZSPA-1 (Eklund et al., 2002) encoding the ZSPA-1 affibody domain, was PCR amplified with Pfu polymerase (Stratagene, La Jolla, CA), using FARO1, 5?GGA ATT CCA TAT GGC TAG CGT AGA CAA ATT CAA CAA AG-3?, as upstream primer and FARO2, 5?-GCG GAT CCA CGG TAC CTT TCG GCG CCT TCG GCG CCT GAG CAT C3?, as downstream primer. Restriction sites for Nhe I and Kpn I were introduced with FARO1 and FARO2, respectively. The amplified ZSPA-1-encoding fragment was cleaved with Kpn I and Nhe I and then ligated into the previously digested pAff8c. Correct insertion was verified by DNA cycle sequencing and analyzed using the MegaBACE

43

system (Amersham Biosciences, Uppsala, Sweden). The resulting novel expression vector was denoted pAff11c.

2.3. Construction of expression vectors Five cDNAs, from a mouse testis library, encoding portions of proteins earlier produced as fusion proteins in the vector pAff8c (Larsson et al., 2000) were selected as model proteins in the evaluation of the novel expression vector system. The five cDNA gene fragments were cleaved out from pAff8c and ligated into pAff11c using the restriction enzymes Asc I and Not I. The constructs, numbered 8, 18, 29, 30 and 54, according to earlier annotations (Larsson et al., 2000), were sequenced on the MegaBACE to confirm correct nucleotide sequence.

2.4. Expression of fusion proteins in pAff11c The five constructs were transformed into E. coli strain BL21(DE3)pLysS. These were grown over night at 37 8C in 250 ml shake flasks containing 25 ml Tryptic Soy Broth (TSB) (Difco, Detroit, MI) (30 g l1) supplemented with yeast extract (5 g l1), 50 mg ml1 kanamycin and 34 mg ml1 chloramphenicol. The cells were sedimented by centrifugation at 1000 /g for 10 min and then resuspended in 20 ml fresh medium. Ten ml of the resuspended cells were inoculated to 500 ml TSB, containing yeast extract and the same antibiotics as above, in 5 l shake flasks. The cultures were incubated at 37 8C until absorption measurements at 600 nm (A600 nm) reached a value of approximately 1.0. Expression was induced by addition of isopropyl-b-D-thiogalactopyranoside (IPTG) (Apollo Scientific Ltd., Whaley Bridge, UK) to a final concentration of 1 mM and the incubation was continued for another 4 h. Samples were collected before induction and at harvest. The cells were harvested by centrifugation at approximately 2000 /g for 10 min and the pellets were resuspended in 30 ml TST buffer (50 mM Tris / HCl, pH 7.5, 2 M NaCl, 0.05% Tween 20) and stored at /20 8C.

44

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

2.5. Affinity purification of the fusion proteins by IMAC The five constructs were also grown in 100 ml, under the same conditions as described above, for purification under denaturing conditions by IMAC, performed essentially as described earlier (Larsson et al., 2000). Briefly, the harvested cells were resuspended in 30 ml lysis buffer (50 mM NaH2PO4, 10 mM Tris /HCl, 6 M Guanidinium / HCl (Gua), 100 mM NaCl, pH 8.0) and stored at /20 8C. The cell suspensions were thawed and sonicated. b-mercaptoethanol was added to a concentration of 10 mM and the cell suspensions were incubated at room temperature on a magnetic stirrer for 1 h. Cell debris was removed by centrifugation and filtration (1.2 mm), and the suspensions were diluted twice with lysis buffer before loading onto 2.5 ml TALON columns (Clontech Laboratories Inc., Palo Alto, CA). Unbound material was washed out with lysis buffer prior to elution of fusion protein with elution buffer (8 M Urea, 100 mM NaCl, 100 mM HAc, 50 mM NaH2PO4, pH 5.0). Relevant fractions were renatured by dialysis against phosphatebuffered saline (PBS) (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM NaCl, pH 7.2).

The protein suspension was loaded onto the column and unbound material was washed out from the column with 30 ml TST prior to elution of the ZSPA-1-fusion proteins with 0.5 M HAc, pH 2.8. Throughout the purification method, a flow rate of 1 ml min 1 was used. Protein contents in the eluted fractions were analyzed by absorption measurements at 280 nm (A280 nm). Relevant fractions were lyophilized and dissolved in PBS. To analyze the protein content in the insoluble fractions, the pellets of the sonicated cell suspensions were analyzed by SDS-PAGE (Phast system, Amersham Biosciences). It was found that pellets from the cultivations of clones 29 and 54 contained high amounts of fusion protein. These pellets were thawed and resuspended in 3 ml 7 M Guanidinium /HCl in TST, supplemented with bmercaptoethanol to a concentration of 3.3 mM. The suspensions were incubated at room temperature on a magnetic stirrer for 1 h. The proteins were renatured by slow dilution with 97 ml TST to a final concentration of approximately 0.2 M Gua. Cell debris was removed by centrifugation and the supernatants were filtered (0.45 mm). The refolded proteins were purified on a 1 ml HiTrap rProtein A column (Amersam Biosciences) under the same conditions as previously described for the soluble ZSPA-1-fusions.

2.6. Affinity purification of the fusion proteins on protein A-Sepharose

2.7. SDS-PAGE and affinity blotting procedures

Cell suspensions were thawed and sonicated (Murby et al., 1995). Cell debris was sedimented by centrifugation at approximately 12 000/g for 10 min and the supernatants, containing the soluble proteins, were filtered (0.45 mm). The pellets, containing proteins that have precipitated as inclusion bodies, were stored at /20 8C. The soluble proteins were purified on protein ASepharose columns (HiTrapTM rProtein A, 1 ml, Amersham Biosciences). The columns were oper¨ KTA ated by the liquid chromatography system, A Explorer (Amersham Biosciences), and a method for purification was created in UNICORN 3.00 (Amersham Biosciences). The fusion proteins were affinity purified with the following method. The column was pulsed three times with TST and 0.5 M HAc, pH 2.8, and then equilibrated with TST.

Whole cell extracts from samples collected before induction and at harvest for each of the cDNA clones, were separated on 10/20% Tris Glycine SDS-PAGE gels (Novex, Invitrogen, Carlsbad, CA). One gel was stained with Coomassie Brilliant Blue and a second gel was electroblotted onto a nitrocellulose membrane (Novex) according to the supplier’s recommendations. The membrane was incubated with 1% milk proteins (Semper, Stockholm, Sweden) in PBS for 45 min. Excess milk proteins were washed away by PBS containing 0.05% Tween 20 (PBST) and the membrane was incubated with 5.2 mg ml 1 protein A (expressed in the vector pk4EDABC, Peter Nilsson, Royal Institute of Technology (KTH), Stockholm, Sweden) for 30 min at room temperature. After a second washing step with PBST, the

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

membrane was incubated with 15 ml peroxidaseanti-peroxidase (PAP) (Sigma) in 15 ml PBS, for 30 min. After a final washing with PBST, the membrane was developed by addition of 3,3diaminobenzidine (Sigma) and 10 ml perhydrol in 10 ml PBS. Whole cell extracts, from the same cultures as above, were also analyzed by a second method. The samples were separated on a 10 /20% Tris / Glycine SDS-PAGE gel (Novex) and electroblotted onto a nitrocellulose membrane (Novex). The membrane was blocked in 1% milk solution for 45 min. Excess milk proteins were washed away with PBST before incubation with 4 mg ml 1 protein A-alkaline phosphatase (PAAP) fusion protein for 30 min. The PAAP fusion protein, consisting of a five-domain native protein A fused in frame with alkaline phosphatase, and encoded by the vector pACEP32 (Sophia Hober, Royal Institute of Technology (KTH), Stockholm, Sweden, unpublished results) was produced by cultivation of RR1DM15 cells harboring the plasmid, in 100 ml TSB
/Y, supplemented with ampicillin (200 mg ml1). Affinity purification of the PAAP fusion protein was performed on IgG /Sepharose (Amersham Biosciences) essentially as described before (Sta˚hl et al., 1999a). The membrane was washed with PBST before development with 5bromo-4-chloro-3-indolyl phosphate /nitro blue tetrazolium (BCIP/NBT) tablets (Sigma) according to the supplier’s recommendations. SDS-PAGE was used to analyze expression levels, purity and stability of the purified fusion proteins. Both protein A-Sepharose- and IMACpurified proteins were separated on 10/20% Tris / Glycine SDS-PAGE gels (Novex). One gel with each set of proteins was stained with Coomassie Brilliant Blue and a second gel was blotted with the PAAP fusion protein as described above. 2.8. Evaluating affinity purification of insoluble fusion proteins on protein A-Sepharose under partly denaturing conditions Clones number 29 and 54, the two least soluble of the five selected cDNA-encoded proteins, were produced a second time to investigate the possibility to recover insoluble fusion proteins in a more

45

straightforward approach. The two constructs were cultivated in 500 ml-scale and harvested as described above. Cell pellets were resuspended in 4 ml TST buffer containing 6 M Gua and stored at /20 8C. Cell suspensions were thawed, sonicated and incubated on a magnetic stirrer in room temperature for 1 h. The denatured proteins were divided in four parts and renatured by slow dilution with TST buffer under continuous stirring to final Gua concentrations of 0.5, 1.0, 1.5 and 2.0 M, respectively. The protein mixtures were incubated at 4 8C with stirring for 1 h. Cell debris was removed by centrifugation and the supernatants were filtered (0.45 mm) before loading onto protein A-Sepharose columns. The affinity purifications were performed on 0.5 ml protein A-Sepharose columns (rProteinA Fast Flow, Amersham Biosciences) at 4 8C. All buffers used had the same Gua concentration in all steps as the respective protein solution to be purified. The columns were equilibrated with 10 ml TST-Gua buffer. Unbound material was washed out from the columns with 10 ml TST-Gua buffer and the fusion proteins were eluted with 0.5 M HAc-Gua, pH 2.8. Protein contents were monitored by adsorption measurements at 280 nm (A280 nm) and relevant fractions were dialyzed against 0.5 M HAc, pH 2.8, before lyophilization. Recovered proteins were analyzed by SDS-PAGE (Phast system, Amersham Biosciences).

3. Results 3.1. Expression vector construction A plasmid vector, pAff11c, was constructed (Fig. 1) for intracellular expression in E. coli of target proteins fused to a dual affinity tag consisting of a His6 sequence and ZSPA-1, a 58 amino acid affibody created by combinatorial protein engineering of a SPA domain (Eklund et al., 2002). The ZSPA-1 affibody shows specific binding to SPA, with a dissociation constant (KD) in the micromolar range (Eklund et al., 2002). The binding of the ZSPA-1 affibody to its parental structure was shown to involve the Fc binding site of SPA (Eklund et al., 2002). The inclusion of

46

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

Fig. 1. The pAff11c vector. A schematic representation of the pAff11c expression vector, designed with an N-terminal dual affinity tag, consisting of a His6 tag and ZSPA-1, a Z affibody with a specific binding capacity to protein A. Expression is tightly regulated by the (PT7)/lac operator (lacO ) sequence located upstream of the sequence encoding the affinity tags. Also encoded by the vector is the lac repressor (LacI) for tight repression prior to induction with IPTG, and the gene conferring resistance to kanamycin (Kmr). The vector also carries the origin of replication for E. coli (pBR322 colE1 ori) and the phage f1 intergenic region.

the ZSPA-1 affibody as part of a His6 /ZSPA-1 dual affinity tag thus allows for affinity purification of expressed fusion proteins to be performed either with IMAC using the His6 tag or on protein ASepharose using the ZSPA-1 tag. The transcription is controlled by the tightly regulated phage T7 promoter (PT7; Studier et al., 1990). 3.2. Protein expression and blotting procedures In order to evaluate the novel expression vector pAff11c, five cDNA clones from a mouse testis cDNA library were selected (Table 1) for expression studies. These cDNA clones had earlier been expressed in the vector pAff8c (Larsson et al., 2000), with yields in the range 20 /50 mg l 1 culture. The five selected cDNA clones, denoted 8, 18, 29, 30 and 54 according to previous nomenclature (Larsson et al., 2000), were restricted from their respective pAff8c constructs and subcloned into the pAff11c expression vector.

The cell cultures for the selected clones were analyzed by SDS-PAGE (Fig. 2A) at the time of induction of protein expression (lanes I), and at the time of cell harvest (lanes H). For each of the five clones it was possible to visually detect the protein band corresponding to the fusion protein in the culture sample collected at harvest only (Fig. 2A), indicating that the expression could be tightly controlled. In the further analysis, a two-step affinityblotting procedure (Fig. 2B), taking advantage of the ZSPA-1-tag present in the expressed fusion proteins, was first evaluated for screening of expression levels directly on cell culture samples. The SDS-PAGE gel (Fig. 2A) was used in a blotting procedure employing recombinant protein A binding to the ZSPA-1 portion of the fusion proteins, as primary reagent, and a PAP complex, consisting of rabbit anti-horseradish peroxidase / IgG complexed with horseradish peroxidase (binding to protein A via the IgG Fc interaction) (Sta˚hl et al., 1999b), as secondary reagent. Tight regulation of the expression was indeed demonstrated since no gene products could be detected in the blots of whole cell lysates at the time of induction (Fig. 2B, lanes I). In contrast, protein bands were readily detected in whole cell lysates from all five cultivations at the time of harvest (Fig. 2B, lanes H), indicating that all cDNA products had been expressed. Secondly, a more convenient single-step affinityblotting procedure was developed using a PAAP fusion protein (Fig. 2C). In this procedure, blotted material was allowed to react with PAAP fusion protein and followed by staining by the addition of alkaline phosphatase substrate. A result similar to that obtained with the two-step affinity blotting procedure was obtained, indicating that this alternative method was similar in sensitivity and selectivity. 3.3. Affinity recovery procedures Affinity purification of the produced proteins was performed both using IMAC employing the His6 fusion tag under denaturing conditions and on protein A-Sepharose using the ZSPA-1 tag. The IMAC-purified proteins were analyzed by SDS-

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

47

Table 1 Clones expressed in pAff11c Clone numbera

Accession numberb

Annotationc

Insert (bp)

Molecular weight (kDa)d

Expression level in pAff11c (mg l 1)

8 18 29 30 54

Z31029 Z31337 Z31105 J02930 Z31156

Outer dense fiber protein Actin-filament associated prot.

650 900 900 900 900

31 38 38 38 38

75 29 50 21 75

a b c d

Laminin B2 Microtubuli-associated prot.

cDNA clones selected from a mouse testis cDNA library. EMBL accession number. As determined from homologies found in public databases. Estimated theoretical mass for the fusion proteins expressed in pAff11c.

PAGE (Fig. 3A), and bands with sizes corresponding to those expected for the full-length target fusion proteins (Table 1) were found to be predominant. However, several smaller bands were detected for some of the clones (especially clone 18). Single-step affinity blotting using the PAAP fusion protein (Fig. 3B) demonstrated that several of the smaller bands were not stained, indicating that these proteins probably were not the result of proteolysis of the target protein, but rather corresponding to other unrelated proteins that have been co-purified using the IMAC procedure. Fusion proteins purified on protein A-Sepharose were also analyzed by SDS-PAGE (Fig. 4A).

Here, a lesser amount of smaller proteins were detected than for the IMAC-purified proteins (Fig. 3A), except for some very small products for clones 8 and 30 (Fig. 4A, lanes 8 and 30), indicating a somewhat more stringent affinity purification procedure. Note, that clones 29 and 54 were predominantly found as inclusion bodies and could be efficiently recovered from insoluble material after denaturation and renaturation, by affinity purification in the presence of 0.2 M guanidinium hydrochloride (Gua). The fusion proteins affinity purified on protein A-Sepharose were also analyzed by affinity blotting using the PAAP fusion protein, and except for clone 29 (Fig. 4A, lane 29), only the major bands were stained.

Fig. 2. Expression of five cDNAs. (A) Five cDNAs, numbered 8, 18, 29, 30 and 54, were inserted in the pAff11c vector and expressed. Whole cell lysates from the cultivations were analyzed by SDS-PAGE under reducing conditions. Lanes labeled I are samples taken out at the time of induction and lanes labeled H are samples taken at harvest. The lane labeled M is a protein size marker with molecular weights of 97, 67, 43, 30, 20 and 14 kDa, respectively. The whole cell lysates were also analyzed by a two-step affinity blotting procedure as shown in (B). After electroblotting of the SDS-PAGE gel to a nitrocellulose membrane, protein A was added, followed by an addition of PAP antibodies. The blot was developed by addition of peroxidase substrate enabling staining of protein bands corresponding to ZSPA-1 fusions (lanes defined as in A). A single-step affinity blotting procedure was also performed as shown in (C). Here, the nitrocellulose membrane was incubated with a PAAP fusion protein. The blot was developed with a phosphatase substrate to enable staining of the protein bands containing ZSPA-1 (lanes defined as in A).

48

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

Fig. 3. (A) The five fusion proteins 8, 18, 29, 30 and 54 were affinity recovered by IMAC, employing the His6 tag, and analyzed by SDS-PAGE under reducing conditions. Lanes labeled M are protein size markers with molecular weights of 97, 67, 43, 30, 20 and 14 kDa, respectively. (B) The IMACpurified proteins were also analyzed by the single-step blotting procedure with PAAP as described in the figure legend for Fig. 2C.

Fig. 4. (A) The five fusion proteins 8, 18, 29, 30 and 54 were affinity recovered on protein A-Sepharose, employing the ZSPA-1 tag, and analyzed by SDS-PAGE under reducing conditions. Bands of sizes corresponding to those expected of the fusion proteins dominate the lanes, but for some, a couple of smaller bands are stained as well. Lanes labeled M are protein size markers with molecular masses of 97, 67, 43, 30, 20 and 14 kDa, respectively. (B) The protein A-Sepharose purified proteins were also analyzed by the single-step blotting procedure using the PAAP fusion protein as described in the figure legend for Fig. 2C.

Expression levels were estimated after affinity purification and all constructs yielded high amounts of protein, ranging from 20 to 75 mg l 1, which were in accordance with expression of the same cDNA-encoded proteins in a different expression system (Larsson et al., 2000). There was no significant difference in the efficiency in recovery between the two different purification methods. In the used experimental set up, the recovery seemed to be very close to 100% for both methods, since the amount of fusion protein found in the

flow through fractions were on the limit of detection (data not shown). Since the protein A-based recovery gave a higher degree of purity, it was considered to be of interest to investigate if an alternative more general recovery concept, independent of the target protein solubility, could be developed based on this affinity tag system. In this, a total cell pellet containing both soluble and insoluble gene products was to be lysed by denaturation, before recovery of the target protein. However, a prerequisite would be that the chaotropic agent used for denaturation does not perturb the interaction between the ZSPA-1 affinity tag and the immobilized protein A ligand. To investigate this parameter, denatured (6 M Gua) cells corresponding to the clones 29 and 54 were divided in four parts and subsequently renatured to four different concentrations of Gua; 0.5, 1.0, 1.5 and 2 M, respectively, before application onto protein A-columns for recovery. Interestingly, fusion proteins were efficiently recovered with 0.5 M Gua, and with as much as 1 M Gua in the buffers specific recovery was still obtained but significantly less efficiently (data not shown). This indicates that the two proteinaceous binding partners ZSPA-1 and protein A are robust and that the interaction between them remains selective also at the unphysiological conditions investigated. Using higher concentrations of Gua (1.5 and 2 M), significant losses were observed. However, the results suggest that a method using direct denaturation of the harvested cells followed by renaturation to 0.5 /1 M Gua prior to the affinity purification procedure, should be highly interesting to evaluate for its general applicability. A final desalting step using either dialysis or a desalting column would be performed to yield the proteins in non-denaturing conditions.

4. Discussion A novel expression vector system has been developed, taking advantage of a 58 amino acid protein A-binding affinity tag ZSPA-1, created by combinatorial protein engineering of a protein A domain, thus enabling straightforward affinity

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and media. Thus, the presented concept combines the benefits of the positive properties of a protein A-based fusion partner, with the robustness and availability of protein A-based chromatographic resins, thus avoiding the drawbacks of IgG-based resins. Protein A-based chromatographic media is the predominant protein-based affinity ligand used in industrial scale bioprocesses, i.e. for affinity recovery of monoclonal antibodies (Fahrner et al., 1999; Tavares et al., 1999). The expression vector was constructed with an N-terminal dual affinity tag, His6 /ZSPA-1, to allow for two alternative affinity recovery principles. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The first affinity blotting procedure includes a two-step concept, in which protein A first is allowed to bind to the blotted fusion proteins containing the ZSPA-1 moiety. Subsequently, a PAP complex is allowed to bind to protein A and the protein bands are stained upon addition of substrate. Secondly, a single-step affinity-blotting procedure was developed using a PAAP fusion protein, which could bind directly to the ZSPA-1 domain, for subsequent staining by addition of substrate. Both affinity blotting procedures did demonstrate tight regulation of the expression which is obviously a crucial feature of a robust expression system also of toxic gene products. The two-step system has the main advantage that protein A and the PAP complex are inexpensive commercially available reagents, while the single-step blotting procedure involving the PAAP fusion protein requires less manual lab work, but with the drawback that the PAAP fusion protein is not commercially available. The five produced fusion proteins were for comparison recovered on both IMAC and protein A columns, and it was found that the protein A-based affinity recovery resulted in the more selective recovery. In this study, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on protein A-Sepharose

49

in the presence of 0.5 M guanidine hydrochloride (data not shown). These results would thus suggest that the presented expression system could be suitable for use as a general high-throughput expression/recovery system for collections of cDNAs with unknown characteristics. A general recovery principle would thus be applicable, including lysis and denaturing of the total cell pellet followed by affinity recovery under partially denaturing conditions, irrespective of the solubility properties of the expressed fusion proteins.

Acknowledgements We thank Christer Ho¨o¨g for creative advice and valuable discussions, Sophia Hober for providing the expression vector encoding the PAAP fusion protein, and Peter Nilsson for the vector encoding protein A. This work has been financially supported by grants from the Cell Factory for Functional Genomics programme from the Foundation for Strategic Research.

References Anderson, N.G., Matheson, A., Anderson, N.L., 2001. Back to the future: the human protein index (HPI) and the agenda for post-genomic biology. Proteomics 1, 3 /12. ˚ ., 2002. Eklund, M., Axelsson, L., Uhle´n, M., Nygren, P.-A Anti-idiotypic protein domains selected from Protein Abased affibody libraries. Proteins: structure, function and genetics, in press. Fahrner, R.L., Whitney, D.H., Vanderlaan, M., Blank, G.S., 1999. Performance comparison of protein A affinity / chromatography sorbents for purifying recombinant monoclonal antibodies. Biotechnol. Appl. Biochem. 30, 121 /128. ˚ ., 1999a. Gunneriusson, E., Nord, K., Uhle´n, M., Nygren, P.-A Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling. Protein Eng. 12, 873 /878. Gunneriusson, E., Samuelson, P., Ringdahl, J., Gro¨nlund, H., ˚ ., Sta˚hl, S., 1999b. Staphylococcal surface Nygren, P.-A display of immunoglobulin A (IgA)- and IgE-specific in vitro-selected binding proteins (affibodies) based on Staphylococcus aureus protein A. Appl. Environ. Microbiol. 65, 4134 /4140. Hansson, M., Sta˚hl, S., Hjorth, R., Uhle´n, M., Moks, T., 1994. Single-step recovery of a secreted recombinant protein by expanded bed adsorption. Bio/Technology 12, 285 /288. Hansson, M., Ringdahl, J., Robert, A., Power, U., Goetsch, L., ˚ ., 1999. Nguyen, T.N., Uhle´n, M., Sta˚hl, S., Nygren, P.-A

50

S. Gra¨slund et al. / Journal of Biotechnology 99 (2002) 41 /50

An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein. Immunotechnology 4, 237 /252. Hopp, T.H., Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, D.P., Urdal, D.L., Conlon, P.J., 1988. A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204 / 1210. Jendeberg, L., Tashiro, M., Tejero, R., Lyons, B.A., Uhle´n, M., Montelione, G.T., Nilsson, B., 1996. The mechanism of binding staphylococcal protein A to immunoglobin G does not involve helix unwinding. Biochemistry 35, 22 /31. Larsson, M., Gra¨slund, S., Yuan, L., Brundell, E., Uhle´n, M., Ho¨o¨g, C., Sta˚hl, S., 2000. High-throughput protein expression of cDNA products as a tool in functional genomics. J. Biotechnol. 80, 143 /157. Murby, M., Samuelsson, E., Nguyen, T.N., Mignard, L., Power, U., Binz, H., Uhle´n, M., Sta˚hl, S., 1995. Hydrophobicity engineering to increase solubility and stability of a recombinant protein from respiratory syncytial virus. Eur. J. Biochem. 230, 38 /44. ˚ ., Nilsson, J., Sta˚hl, S., Lundeberg, J., Uhle´n, M., Nygren, P.-A 1997. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11, 1 /16. ˚ ., Nord, K., Nilsson, J., Nilsson, B., Uhle´n, M., Nygren, P.-A 1995. A combinatorial library of an a-helical bacterial receptor domain. Protein Eng. 8, 601 /608. Nord, K., Gunneriusson, E., Ringdahl, J., Sta˚hl, S., Uhle´n, M., ˚ ., 1997. Binding proteins selected from combiNygren, P.-A natorial libraries of an a-helical bacterial receptor domain. Nat. Biotechnol. 15, 772 /777. ˚ ., 2000. Nord, K., Gunneriusson, E., Uhle´n, M., Nygren, P.-A Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1M and taq DNA polymerase. J. Biotechnol. 80, 45 /54. Nord, K., Nord, O., Uhle´n, M., Kelley, B., Ljungqvist, C., ˚ ., 2001. Recombinant human factor VIIINygren, P.-A

specific affinity ligands selected from phage-displayed combinatorial libraries of protein A. Eur. J. Biochem. 268, 4269 /4277. Porath, J., Carlsson, J., Olsson, I., Belfrage, G., 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598 /599. Ru¨ther, U., 1982. pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts. Nucleic Acids Res. 10, 5765 /5772. Simons, P.C., Vander Jagt, D.L., 1981. Purification of glutathione S-transferases by glutathione-affinity chromatography. Methods Enzymol. 77, 235 /237. Smith, D.B., Johnson, K.S., 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31 /40. Studier, F.W., Rosenberg, A.H., Dunn, J.J., Dubendorff, J.W., 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60 /89. ˚ ., Sta˚hl, S., Nilsson, J., Hober, S., Uhle´n, M., Nygren, P.-A 1999a. Affinity fusions, gene expression. In: Flickinger, M.C., Drew, S.W. (Eds.), Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. Wiley, New York, pp. 49 /63. Sta˚hl, S., Odeberg, J., Larsson, M., Rosok, O., Ree, A.H., Lundeberg, J., 1999b. Solid-phase differential display and bacterial expression systems in selection and functional analysis of cDNAs. Methods Enzymol. 303, 495 /511. Tavares, R.M., Karmali, A., Clemente, A., Pais, M.S., 1999. Production and characterization of a specific rubisco monoclonal antibody, and its use in rubisco quantification during Zantedeschia aethiopica spathe development. Hybridoma 18, 203 /209. Uhle´n, M., Nilsson, B., Guss, B., Lindberg, M., Gatenbeck, S., Philipson, L., 1983. Gene fusion vectors based on the gene for staphylococcal protein A. Gene 23, 369 /378. Uhle´n, M., Forsberg, G., Moks, T., Hartmanis, M., Nilsson, B., 1992. Fusion proteins in biotechnology. Curr. Opin. Biotechnol. 3, 363 /369.