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Expression and purification of cysteine introduced recombinant saporin
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Protein Expression and Purification 58 (2008) 203–209 www.elsevier.com/locate/yprep
Expression and purification of cysteine introduced recombinant saporin Emine Gu¨nhan a
a,*
, Mimi Swe a, Mine Palazoglu a, John C. Voss b, Leo M. Chalupa
a
Department of Neurobiology, Physiology, and Behavior, University of California, One Shields Avenue, 196 Briggs Hall, Davis, CA 95616, USA b Department of Biochemistry, University of California, Davis, CA 95616, USA Received 11 September 2007, and in revised form 8 November 2007 Available online 19 November 2007
Abstract Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC50 values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity. 2007 Elsevier Inc. All rights reserved. Keywords: Saporin; Immunotoxin
Introduction Immunotoxins, chimeric molecules containing a monoclonal antibody or its fragment conjugated to a toxin [1], are often termed ‘‘magic bullets’’ [2] because of their specific targeted toxicities. Immunotoxin specificity stems from their cell binding fragments and their potency from the toxins conjugated to these fragments. Immunotoxins have found many applications in neuroscience research [3–5]. In the last decade, more than 1500 research articles have been published about immunotoxins and more than 10% of these were related to neuroscience research. Ribosome inactivating proteins (RIPs)1 are one group of toxins widely used in immunotoxin production. RIPs are N-glycosidases that recognize and remove a single adenine residue located in a highly conserved region of 28S ribo*
Corresponding author. Fax: +1 530 752 8908. E-mail address: [email protected] (E. Gu¨nhan). 1 Abbreviations used: RIP, ribosome inactivating proteins; sap, saporin; EF, elongation factor; SPDP, N-succynimidyl 3-(2-pyridyl-dithiopropionate; TCEP HCl, tris (2-carboxyethyl) phosphine hydrochloride. 1046-5928/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2007.11.005
somal RNA [6,7]. Because this particular adenine residue engages in binding elongation factor (EF)-1 and EF-2 to the ribosome [8], RIPs prevent the elongation step in protein synthesis. There are two types of RIPs. Type I RIPs are single-chain proteins while type II RIPs have two polypeptide chains linked by disulfide bonds [9]. RIPs conjugated to a cell binding vector (e.g. monoclonal antibodies) target specific cells. When the targeted cells internalize an active chain of RIPs the end result is cell death by apoptosis [10,11]. RIPs have great therapeutic potentials as chimeric toxins such as immunotoxins, for treating cancer and autoimmune diseases [12–14]. Saporin, a type I RIP with a molecular weight of 30 kDa, is extracted from the seeds of Saponaria officinalis L. Saporin destroys both eukaryotic and prokaryotic ribosomes [15–17]. There are five isoforms of saporin isolated from S. officinalis L., four are from the seed of the plant (isoforms 1, 3, 4 and 6) [18] and one is isolated from the leaf of the plant [19,20]. Indeed, several different genomic clones have been identified, confirming the existence of a multigene saporin family [21]. Saporin has a passive and low cell binding affinity [22] which decreases nonspecific
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cell binding [22,23]. In addition, saporin is very stable and keeps its enzymatic activity even under harsh conditions such as denaturation and proteolysis [24]. Because of these two important properties, saporin is widely used as the potent toxin counterpart of immunotoxins in neuroscience and other applications [23]. Type 1 (single-chain) RIPs become cytotoxic only when the toxin molecule is separated from the carrier molecule in an active form. The separation process is an important step since it is necessary to avoid steric hindrance and allows an effective translocation of the toxin into the cytoplasm [25]. This can be accomplished by disulfide bonding [26] for the coupling of the toxin to a carrier molecule [9]. The most common way to modify proteins is by chemical introduction of thiol groups using heterobifunctional reagents, for example, SPDP (N-succynimidyl 3-(2-pyridyl-dithiopropionate). SPDP has amine and sulfhydryl reactive groups. Amine-reactive group of SPDP, N-hydrosuccinimide ester reacts with the primary amine on the protein; at the other end, 2-pyridyldithio group will react with the sulfhydryl group on the second protein, forming a covalent link between the two proteins. A simpler means of modifying proteins is by targeting cysteine molecules in the protein molecules since cysteine occurs at low frequency in protein sequences and provides a highly reactive sulfhydryl group. Since type 1 RIPs do not contain native cysteine residues in their sequences, specificity of conjugation within the protein sequence can be readily achieved. In our study, sap-3 isomer (a kind gift from S. Fabbrini) was mutated at the carboxyl terminal of sap-3 gene. Cysteine mutation was introduced by antisense primer utilizing PCR, and the new product was named as Cys255sap-3. IC50 values in cell-free systems for both Cys255sap-3 and sap-3 were found to be similar. We found that Cys255sap-3 reacts with high efficiency to the pyridyldithio moiety of SPDP-labeled antibody, providing a convenient route for attaching saporin to the antibody in its active form. Materials and methods The plasmid construction, transformation, DNA extraction and production of the cell bank are explained in detail below. After verification of the mutation; a master cell bank was established. The cell bank was always used as a starter culture for Cys255sap-3 protein expression. Cloning of recombinant saporin in pET11d plasmid We used the sap-3 isomer, a 253 amino acid long protein, to introduce cysteine into its sequence. We designed sense and antisense oligos by using Vector NTI program. We introduced the cysteine mutation at the C-terminus of the sap-3 gene, at position 255 by antisense primer utilizing polymerase chain reaction. Serine at position 255 was replaced by cysteine. The choice of the mutation site was
based on two considerations. First, we found that the cysteine residue at the N-terminal resulted in unstable mutations (our unpublished observations). Second, cysteine needed to be far apart from the ribosome inactivating site residues, (Tyr16, Arg24, Tyr72, Tyr120, Glu176, Arg179 and Trp208) [16,27] to ensure that the enzymatic activity of the toxin remains intact. The new product was named as Cys255sap-3. NcoI and EcoRI enzyme restriction sites were preserved at the ends of the recombinant saporin gene, allowing cloning into pET11d plasmid using the same cloning strategy as Fabbrini et al. [15]. This eliminated the T7 terminator on the plasmid; but did not have an appreciable effect on protein expression yield. Enzyme digest with NcoI and EcoRI revealed two bands on the agarose gel, one band at 0.8 kb Cys255sap-3 gene and the other band at 5.5 kb pET11d. Once the presence of the two distinct bands was confirmed, plasmid Cys255sap-3 gene was sequenced to ensure that the mutation had occurred and had ligated into the correct reading frame of the pET11d plasmid. The efficiency of the transformation and success of the cysteine mutation were verified by extracting DNA from overnight cultures of the Escherichia coli hosts. Transformation by heat shock, and extraction of plasmid DNA We used two different E. coli host strains for the production of Cys255sap-3; XL-1 Blue and BL21 (DE3) pLysS E. coli strains. XL-1 Blue cells, designed for DNA manipulations, have high cloning efficiency. They also have high efficiency in transformation, leading to high Cys255sap-3 DNA concentrations in overnight cultures, allowing more accurate DNA sequencing results. On the other hand, BL21 (DE3) pLysS E. coli strain yields high protein expression of the recombinant saporin. pET11d plasmid was transformed into XL-1 Blue Supercompetent Cells, following manufacturer’s protocol for heat shock (Stratagene) and into BL21 (DE3) pLysS Competent Cells (Novagen). Respective transformations were plated onto LB-agar plates containing 100 lg/ml ampicillin and incubated at 37 C incubator. The efficiency of transformations and the success of cysteine mutations were verified by extracting DNA from respective E. coli host overnight cultures. Enzyme digestion was performed using NcoI and EcoRI enzymes. The presence of the saporin gene and pET11d vector (0.753 kb, 5.3 kb, respectively) was verified on a gel. DNA sequencing was performed by a commercial sequencing laboratory by using SI forward primer. Vector NTI program was used to verify that the cysteine mutation was introduced at the right position. Cell bank Master cell bank was established after verifying the mutation. A single colony was inoculated into 5 ml LB
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Broth supplemented with 50 lg/ml ampicillin and grown to an OD600 of 0.8. Cells were collected by centrifugation and resuspended in 1 ml LB Broth containing 8% glycerol and stored at 80 C. Protein expression of Cys255sap-3 and purification The transformed bacteria from the master cell bank were inoculated into LB broth fortified with 100 lg/ml ampicillin. Cultures were grown overnight at 37 C in a New Brunswick incubator to an OD600 of 4.0–4.5. Overnight cultures (6 · 10 ml) were added to 340 ml LB Broth fortified with 100 lg/ml ampicillin. When the OD600 value is 0.6–0.8, protein expression was induced by adding 1 mM IPTG to 400 ml culture. Incubation was continued under rigorous shaking at 37 C for 4 h. Cells were harvested at 10,000g for 30 min (Beckman centrifuge, JA-14 rotor). A volume of 5 ml of BugBuster supplemented by 2 mM DTT were added (to prevent dimerization via disulfide bond forming between the engineered cysteine residues). A concentration of 5 mM EDTA was added for each cell pellet (obtained from 400 ml culture, weighing about 1 g) to resuspend cells. The resuspended cells were incubated at room temperature on a shaker for 20 min and pelleted at 16,000g for 30 min at 4 C. Solutions were filtered through 0.22 micron filters. Filtered soluble protein lysate was dialyzed against 1 L 25 mM sodium chloride in pH 6.0 5 mM phosphate buffer with 2 mM EDTA for 4 h, changing the dialysis buffer every 2 h. Capturing and purification steps were achieved by using HP CM FF Sepharose ion exchange columns (GE Healthcare). These columns were found to be suitable for our application and gave good purification yields. Conjugation of Cys255sap-3 to a fluorescent dye Cys255sap-3 was conjugated to Alexa Fluor 568 C5 maleimide (Invitrogen, CA). A volume of 100 ll of Cys255sap-3 solution (1 mg/ml in PBS with 2 mM EDTA) and mixed with 2 ll of Alexa Fluor 568 C5 maleimide (10 mM). The mixture was incubated at room temperature for 45 min. The conjugate was separated from the unconjugated dye using Bio-Spin 6 Tris columns (Bio-Rad, CA). Conjugation of Cys255sap-3 to a nonspecific IgG A quantity of 0.5 mg of mouse anti-human IgG (5 mg/ ml, Jackson Immuno, MA) was mixed with 5 lg SPDP (Pierce Biotechnology, IL) in reaction buffer (0.04 M sodium phosphate, 0.0150 M sodium chloride, pH 7.5) and incubated at room temperature for 1 h. To remove unreacted cross-linker, buffer was exchanged with fresh reaction buffer. 0.5 mg of Cys255sap-3 (1 mg/ml) was added to the mixture and incubated for 1 h at RT and 17 h at 4 C. Free Cys255sap-3 was removed using Protein G columns (Pierce Biotechnology, IL).
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Protein synthesis inhibition assay Enzymatic activities of recombinant Cys255sap-3 and Cys255sap-3 conjugated to nonspecific IgG were evaluated by protein synthesis inhibition. Serial log dilutions starting from 3.3 lM to 3 pM were made in sterile RNase and DNase free phosphate buffered saline. Two separate experiments were made and all dilutions were read in triplicates. Samples were read on a luminator assay using Luciferase Assay Reagent (Promega corporation, WI). Data analysis was performed by using MatLab. Results and discussion Ribosome inactivating proteins (RIPs) are valuable therapeutic and research agents. Their utility derives from their ability to interact with ribosomes and stop protein synthesis, resulting in cell death. RIPs conjugated to molecules (e.g. antibodies, cytokines, peptides) have been used to target specific cell populations [28]. Because of this, RIPs are widely used for targeted biologic therapy in diseases such as cancer and autoimmune diseases [12–14,29,30]. The approach underlying these therapies is to exploit cell specific surface antigens. RIPs are unique in their ability to kill a cell with a single molecule [31,32], due to their extreme potency. Cell death results when, after internalization, a catalytic fragment of the toxin enters the cytosol [25]. Nonspecific conjugation chemistry of RIPs to targeting peptides or antibodies is usually accompanied by a decrease in the pharmacokinetic properties of the conjugate. One of the most common methods to couple two protein molecules together is to employ a heterobifunctional cross-linking agent whose functional moieties react with specific side chain groups in proteins (e.g. amine groups, sulfhydryl groups). Achieving specificity is a major challenge to this approach, however. For example, amine groups (e.g. lysine residues) occur in high frequency throughout protein sequences. Therefore, conjugation usually occurs randomly throughout the protein structure when amine-reactive agents are employed, often compromising the natural properties of these residues (e.g. ribosome inactivation). Conjugation to a specific residue provides the advantage of a predictable and reproducible structure. In this regard, use of cysteine side chains for targeting cross-linking reagents is advantageous. Cysteine residues are found at lower frequencies within protein sequences, and the conjugation reaction is more efficient using the highly reactive sulfhydryl group of the cysteine. Type 1 RIPs do not contain cysteine residues in their sequences. Saporin extracted from S. officinalis L. is one of the most commonly used type I RIPs for generating immunotoxins [23]. We have engineered a cysteine residue at position 255 of the sap-3 isomer with the intent of introducing a site for chemical conjugation. This makes the conjugation reaction more efficient and less complicated. Moreover, unlike other protein modification methods by
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which the modification could occur at any part of the saporin molecule, a single cysteine ensures a specific binding site. In our case, the important factors for the choice of the mutation site were: (1) the modification should not affect ribosome binding (i.e. should not be in the catalytic site of the saporin), (2) the engineered site should be easily accessible for conjugation and (3) the new protein should be stable. Previous studies show that all residues in the catalytic active site cleft of RIP proteins are conserved [15] and mutations at these catalytic active site cleft residues cause a drastic loss of activity [33]. In addition, our observations indicate that mutations in the N terminus produce unstable proteins (unpublished). One modification that meets each of these criteria is the introduction of a cysteine residue at position 255 of sap-3 isomer. The engineered recombinant saporin was expressed in BL(21) E. coli cells, where recombinant protein was expressed in the soluble fraction. Expression of the recombinant saporin gene We found that the Cys255sap-3 cloned into the pET11d plasmid achieves suitable expression in BL21 (DE3) pLysS cells, despite the protein’s toxicity. Following induction for protein expression, induced bacteria were lysed, sonicated and fractionated by centrifugation. Soluble and insoluble fractions were then analyzed by SDS–PAGE, followed by Western blot analysis using chicken anti-saporin antibody. The results revealed that Cys255sap-3 is expressed in soluble fraction, with no indication of Cys255sap-3 in the insoluble fraction (not shown). Expression yield was only slightly improved when induction time was extended to 4 h as compared to 2 h. Extraction and purification of the Cys255Sap-3 protein Recombinant sap-3 protein was then isolated using size-exclusion chromatography (Fig. 1). As may be seen, SDS–PAGE gels show a major band with an apparent MW of 30 kDa, slightly higher than sap-3 isomer, a 253 amino acid long protein. This difference in migration is likely due to the extra residues added to the recombinant saporin (260 amino acids, the last 7 amino acids added are, Ala, Cys, Glu, Lys, Asp, Glu and Leu). We also noted a minor impurity (at 26 kDa) that co-purifies with the recombinant saporin. Since this was not detected in the Western blot that utilizes a polyclonal antibody derived against the intact saporin protein, it is unlikely that this smaller band represents a proteolytic fragment of the saporin protein. The gels in Figs. 1–3 were all run under non-reducing conditions. With Coomassie Blue staining, no dimerization via a disulfide bridge involving Cys255 was evident. Thus, this protein provides a site with a low propensity to form intramolecular disulfide bonds, a useful property for sites that will later undergo covalent modification. However, using the more sensitive immunoblot method,
Fig. 1. SDS–PAG E (A) and Western blot (B) analysis of saporin, sap-3 isomer and Cys255sap-3. (A) SDS–PAGE analysis was done with nonreducing SDS gels. Lane 1: protein marker, lane 2: commercial saporin, lanes 3 and 4 Cys255sap-3, lane 5: sap-3 isomer. (B) Western blot assay using the polyclonal anti-saporin antibodies (dilution 0.36 lg/ml). Lane 1: protein marker, lane2: commercial saporin, lane 3: sap-3 isomer, lanes 4 and 5: Cys255sap-3.
a species at approximately twice the size of monomeric saporin was revealed. In summary, the purification described here provides a rapid reproducible and convenient method for obtaining yields of 2.7 mg/l culture (Table 1). The activity of the cysteine residue in the Cys255sap-3 To confirm the reactivity of Cys255 in the sap-3 protein, we first conjugated Cys255sap-3 with a fluorescent dye (Alexa 568 C5 maleimide, Invitrogen, CA), (Fig. 2, left panel) and then stained the same gel with coomassie (Fig. 2, right panel). Prior treatment of the Cys255sap-3 protein with the reducing agent Tris (2-carboxyethyl) phosphine Hydrochloride (TCEP HCl) before conjugating with fluorescent dye did not result in improved labeling levels. There were no visible bands for dimers under non-reducing SDS–PAGE gels (Figs. 1A and 3), but dimers were present in the Western blot (Fig. 1B). Such a difference is often encountered because of the sensitiv-
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Fig. 2. SDS–PAGE analysis of Alexa 568 tagged Cys255sap-3. Left column (lanes 1 and 2), image of non-reducing SDS–PAGE gel scanned on Typhoon Scanner using the 580 nm laser. Right column (lanes 3 and 4) Coomassie stained non-reducing SDS–PAGE gel (the same gel as left column). Lanes 1 and 4 protein marker, lanes 2 and 3: Alexa 568 tagged Cys255Sap-3, (2 lg).
ity differences between the two assays. Not detecting the bands in SDS gels indicates that the amount of dimerization is minimal. Thus, we concluded that Cys255 does not represent a position in the protein that has a high propensity to dimerize via disulfide bands. Such a feature would complicate the efficiency of modifying this site with cross-linking reagents. To validate that Cys255 in the recombinant saporin is suitable for cross-linking to antibody, we conjugated the Cys255sap-3 to a nonspecific mouse IgG. Coomassie stained SDS–PAGE showed that a high level of cross-linking was achieved when the heterobifunctional reagent was targeted to Cys255 in saporin (Fig. 3).
Fig. 3. SDS–PAGE analysis of recombinant saporin, mouse anti-human IgG and recombinant saporin conjugated to mouse anti-human IgG. Lane 1, protein marker, lane 2, Cys255sap-3 (2 lg), lane 3, mouse anti-human IgG (2.4 lg), lane 4, Cys255sap-3 conjugated to mouse anti-human IgG (3.8 lg). Table 1 Total protein and Cys255sap-3 contents at different stages of purification of recombinant Cys255sap-3 Fraction
Total protein amount, measured by BCA assay (per 1 L culture)
Total Cys255sap-3 amount (per 1 L culture)
Approximate yield (%)
Total cell lysate Soluble cell lysate After purification (95% purity)
350 mg 120 mg —
15.6 mg 12.5 mg 2.7 mg
100 80 17
The preparation was with 2.5 g wet weight cells from 1 L of culture.
Ribosome inactivating activity of the Cys255sap-3 Ribosome inactivating activity of Cys255sap-3 expressed in BL21 (DE3) pLysS cells was tested by in vitro translation inhibition assay using rabbit reticulocyte lysates. The enzymatic activity of recombinant Cys255sap-3 was compared to those of sap-3 isomer and commercial saporin purchased from Advanced Targeting Systems. Fig. 4, a semi-log graph, shows the protein synthesis (% control) as a function of concentration where
concentration is expressed in log values. These values (10 12.0744 for Cys255sap-3, 10 11.2978 for sap-3 isomer and 10 11.5014 for commercial saporin) were calculated by MatLab using the experimental data (three replicates at each point) as seen in the graph. IC50 values were than calculated by taking anti-log values of concentration values. The purified Cys255sap-3 displayed a strong inhibitory activity on protein synthesis in the cell-free rabbit
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Fig. 4. Semi-log graph showing the protein synthesis inhibition as a function of toxin concentration. Activities for Cys255sap-3, Sap-3 isomer and commercial saporin were tested by protein synthesis inhibition assay. One microliter of mRNA was added to 0.5 ml RNase-free microcentrifuge tubes and incubated 3 min at 65 C. One microliter of samples (dilutions of Cys255sap-3, Sap-3 isomer and commercial saporin), 7 ll of rabbit reticulocyte lysate (Promega), 1 ll of amino acid minus mixture and 0.2 ll of RNasin were added to respective tubes and incubated at 30 C in a water bath for 30 min. Two microliters of samples from each tube were added on 96-well opaque plate and read on L-Max luminometer using 25 ll Luciferase Assay Reagent (Promega). Data were analyzed using MatLab. Green, Sap-3 isomer; Blue, Cys255sap-3; Red, commercial saporin (purchased from Advanced Targeting Systems).
reticulocyte system, with an IC50 of 0.84 · 10 12. This value is on the order of native sap-3 isomer (5.0 · 10 12) and commercial saporin (3.1 · 10 12) (Fig. 4). In conclusion, Cys255 is a good position to target/place the thiol group because it does not have a high propensity to form intermolecular disulfides, it provides good linker flexibility, and modification at this site does not reduce saporin activity.
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Protein Expression and Purification 58 (2008) 203–209 www.elsevier.com/locate/yprep
Expression and purification of cysteine introduced recombinant saporin Emine Gu¨nhan a
a,*
, Mimi Swe a, Mine Palazoglu a, John C. Voss b, Leo M. Chalupa
a
Department of Neurobiology, Physiology, and Behavior, University of California, One Shields Avenue, 196 Briggs Hall, Davis, CA 95616, USA b Department of Biochemistry, University of California, Davis, CA 95616, USA Received 11 September 2007, and in revised form 8 November 2007 Available online 19 November 2007
Abstract Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC50 values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity. 2007 Elsevier Inc. All rights reserved. Keywords: Saporin; Immunotoxin
Introduction Immunotoxins, chimeric molecules containing a monoclonal antibody or its fragment conjugated to a toxin [1], are often termed ‘‘magic bullets’’ [2] because of their specific targeted toxicities. Immunotoxin specificity stems from their cell binding fragments and their potency from the toxins conjugated to these fragments. Immunotoxins have found many applications in neuroscience research [3–5]. In the last decade, more than 1500 research articles have been published about immunotoxins and more than 10% of these were related to neuroscience research. Ribosome inactivating proteins (RIPs)1 are one group of toxins widely used in immunotoxin production. RIPs are N-glycosidases that recognize and remove a single adenine residue located in a highly conserved region of 28S ribo*
Corresponding author. Fax: +1 530 752 8908. E-mail address: [email protected] (E. Gu¨nhan). 1 Abbreviations used: RIP, ribosome inactivating proteins; sap, saporin; EF, elongation factor; SPDP, N-succynimidyl 3-(2-pyridyl-dithiopropionate; TCEP HCl, tris (2-carboxyethyl) phosphine hydrochloride. 1046-5928/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2007.11.005
somal RNA [6,7]. Because this particular adenine residue engages in binding elongation factor (EF)-1 and EF-2 to the ribosome [8], RIPs prevent the elongation step in protein synthesis. There are two types of RIPs. Type I RIPs are single-chain proteins while type II RIPs have two polypeptide chains linked by disulfide bonds [9]. RIPs conjugated to a cell binding vector (e.g. monoclonal antibodies) target specific cells. When the targeted cells internalize an active chain of RIPs the end result is cell death by apoptosis [10,11]. RIPs have great therapeutic potentials as chimeric toxins such as immunotoxins, for treating cancer and autoimmune diseases [12–14]. Saporin, a type I RIP with a molecular weight of 30 kDa, is extracted from the seeds of Saponaria officinalis L. Saporin destroys both eukaryotic and prokaryotic ribosomes [15–17]. There are five isoforms of saporin isolated from S. officinalis L., four are from the seed of the plant (isoforms 1, 3, 4 and 6) [18] and one is isolated from the leaf of the plant [19,20]. Indeed, several different genomic clones have been identified, confirming the existence of a multigene saporin family [21]. Saporin has a passive and low cell binding affinity [22] which decreases nonspecific
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cell binding [22,23]. In addition, saporin is very stable and keeps its enzymatic activity even under harsh conditions such as denaturation and proteolysis [24]. Because of these two important properties, saporin is widely used as the potent toxin counterpart of immunotoxins in neuroscience and other applications [23]. Type 1 (single-chain) RIPs become cytotoxic only when the toxin molecule is separated from the carrier molecule in an active form. The separation process is an important step since it is necessary to avoid steric hindrance and allows an effective translocation of the toxin into the cytoplasm [25]. This can be accomplished by disulfide bonding [26] for the coupling of the toxin to a carrier molecule [9]. The most common way to modify proteins is by chemical introduction of thiol groups using heterobifunctional reagents, for example, SPDP (N-succynimidyl 3-(2-pyridyl-dithiopropionate). SPDP has amine and sulfhydryl reactive groups. Amine-reactive group of SPDP, N-hydrosuccinimide ester reacts with the primary amine on the protein; at the other end, 2-pyridyldithio group will react with the sulfhydryl group on the second protein, forming a covalent link between the two proteins. A simpler means of modifying proteins is by targeting cysteine molecules in the protein molecules since cysteine occurs at low frequency in protein sequences and provides a highly reactive sulfhydryl group. Since type 1 RIPs do not contain native cysteine residues in their sequences, specificity of conjugation within the protein sequence can be readily achieved. In our study, sap-3 isomer (a kind gift from S. Fabbrini) was mutated at the carboxyl terminal of sap-3 gene. Cysteine mutation was introduced by antisense primer utilizing PCR, and the new product was named as Cys255sap-3. IC50 values in cell-free systems for both Cys255sap-3 and sap-3 were found to be similar. We found that Cys255sap-3 reacts with high efficiency to the pyridyldithio moiety of SPDP-labeled antibody, providing a convenient route for attaching saporin to the antibody in its active form. Materials and methods The plasmid construction, transformation, DNA extraction and production of the cell bank are explained in detail below. After verification of the mutation; a master cell bank was established. The cell bank was always used as a starter culture for Cys255sap-3 protein expression. Cloning of recombinant saporin in pET11d plasmid We used the sap-3 isomer, a 253 amino acid long protein, to introduce cysteine into its sequence. We designed sense and antisense oligos by using Vector NTI program. We introduced the cysteine mutation at the C-terminus of the sap-3 gene, at position 255 by antisense primer utilizing polymerase chain reaction. Serine at position 255 was replaced by cysteine. The choice of the mutation site was
based on two considerations. First, we found that the cysteine residue at the N-terminal resulted in unstable mutations (our unpublished observations). Second, cysteine needed to be far apart from the ribosome inactivating site residues, (Tyr16, Arg24, Tyr72, Tyr120, Glu176, Arg179 and Trp208) [16,27] to ensure that the enzymatic activity of the toxin remains intact. The new product was named as Cys255sap-3. NcoI and EcoRI enzyme restriction sites were preserved at the ends of the recombinant saporin gene, allowing cloning into pET11d plasmid using the same cloning strategy as Fabbrini et al. [15]. This eliminated the T7 terminator on the plasmid; but did not have an appreciable effect on protein expression yield. Enzyme digest with NcoI and EcoRI revealed two bands on the agarose gel, one band at 0.8 kb Cys255sap-3 gene and the other band at 5.5 kb pET11d. Once the presence of the two distinct bands was confirmed, plasmid Cys255sap-3 gene was sequenced to ensure that the mutation had occurred and had ligated into the correct reading frame of the pET11d plasmid. The efficiency of the transformation and success of the cysteine mutation were verified by extracting DNA from overnight cultures of the Escherichia coli hosts. Transformation by heat shock, and extraction of plasmid DNA We used two different E. coli host strains for the production of Cys255sap-3; XL-1 Blue and BL21 (DE3) pLysS E. coli strains. XL-1 Blue cells, designed for DNA manipulations, have high cloning efficiency. They also have high efficiency in transformation, leading to high Cys255sap-3 DNA concentrations in overnight cultures, allowing more accurate DNA sequencing results. On the other hand, BL21 (DE3) pLysS E. coli strain yields high protein expression of the recombinant saporin. pET11d plasmid was transformed into XL-1 Blue Supercompetent Cells, following manufacturer’s protocol for heat shock (Stratagene) and into BL21 (DE3) pLysS Competent Cells (Novagen). Respective transformations were plated onto LB-agar plates containing 100 lg/ml ampicillin and incubated at 37 C incubator. The efficiency of transformations and the success of cysteine mutations were verified by extracting DNA from respective E. coli host overnight cultures. Enzyme digestion was performed using NcoI and EcoRI enzymes. The presence of the saporin gene and pET11d vector (0.753 kb, 5.3 kb, respectively) was verified on a gel. DNA sequencing was performed by a commercial sequencing laboratory by using SI forward primer. Vector NTI program was used to verify that the cysteine mutation was introduced at the right position. Cell bank Master cell bank was established after verifying the mutation. A single colony was inoculated into 5 ml LB
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Broth supplemented with 50 lg/ml ampicillin and grown to an OD600 of 0.8. Cells were collected by centrifugation and resuspended in 1 ml LB Broth containing 8% glycerol and stored at 80 C. Protein expression of Cys255sap-3 and purification The transformed bacteria from the master cell bank were inoculated into LB broth fortified with 100 lg/ml ampicillin. Cultures were grown overnight at 37 C in a New Brunswick incubator to an OD600 of 4.0–4.5. Overnight cultures (6 · 10 ml) were added to 340 ml LB Broth fortified with 100 lg/ml ampicillin. When the OD600 value is 0.6–0.8, protein expression was induced by adding 1 mM IPTG to 400 ml culture. Incubation was continued under rigorous shaking at 37 C for 4 h. Cells were harvested at 10,000g for 30 min (Beckman centrifuge, JA-14 rotor). A volume of 5 ml of BugBuster supplemented by 2 mM DTT were added (to prevent dimerization via disulfide bond forming between the engineered cysteine residues). A concentration of 5 mM EDTA was added for each cell pellet (obtained from 400 ml culture, weighing about 1 g) to resuspend cells. The resuspended cells were incubated at room temperature on a shaker for 20 min and pelleted at 16,000g for 30 min at 4 C. Solutions were filtered through 0.22 micron filters. Filtered soluble protein lysate was dialyzed against 1 L 25 mM sodium chloride in pH 6.0 5 mM phosphate buffer with 2 mM EDTA for 4 h, changing the dialysis buffer every 2 h. Capturing and purification steps were achieved by using HP CM FF Sepharose ion exchange columns (GE Healthcare). These columns were found to be suitable for our application and gave good purification yields. Conjugation of Cys255sap-3 to a fluorescent dye Cys255sap-3 was conjugated to Alexa Fluor 568 C5 maleimide (Invitrogen, CA). A volume of 100 ll of Cys255sap-3 solution (1 mg/ml in PBS with 2 mM EDTA) and mixed with 2 ll of Alexa Fluor 568 C5 maleimide (10 mM). The mixture was incubated at room temperature for 45 min. The conjugate was separated from the unconjugated dye using Bio-Spin 6 Tris columns (Bio-Rad, CA). Conjugation of Cys255sap-3 to a nonspecific IgG A quantity of 0.5 mg of mouse anti-human IgG (5 mg/ ml, Jackson Immuno, MA) was mixed with 5 lg SPDP (Pierce Biotechnology, IL) in reaction buffer (0.04 M sodium phosphate, 0.0150 M sodium chloride, pH 7.5) and incubated at room temperature for 1 h. To remove unreacted cross-linker, buffer was exchanged with fresh reaction buffer. 0.5 mg of Cys255sap-3 (1 mg/ml) was added to the mixture and incubated for 1 h at RT and 17 h at 4 C. Free Cys255sap-3 was removed using Protein G columns (Pierce Biotechnology, IL).
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Protein synthesis inhibition assay Enzymatic activities of recombinant Cys255sap-3 and Cys255sap-3 conjugated to nonspecific IgG were evaluated by protein synthesis inhibition. Serial log dilutions starting from 3.3 lM to 3 pM were made in sterile RNase and DNase free phosphate buffered saline. Two separate experiments were made and all dilutions were read in triplicates. Samples were read on a luminator assay using Luciferase Assay Reagent (Promega corporation, WI). Data analysis was performed by using MatLab. Results and discussion Ribosome inactivating proteins (RIPs) are valuable therapeutic and research agents. Their utility derives from their ability to interact with ribosomes and stop protein synthesis, resulting in cell death. RIPs conjugated to molecules (e.g. antibodies, cytokines, peptides) have been used to target specific cell populations [28]. Because of this, RIPs are widely used for targeted biologic therapy in diseases such as cancer and autoimmune diseases [12–14,29,30]. The approach underlying these therapies is to exploit cell specific surface antigens. RIPs are unique in their ability to kill a cell with a single molecule [31,32], due to their extreme potency. Cell death results when, after internalization, a catalytic fragment of the toxin enters the cytosol [25]. Nonspecific conjugation chemistry of RIPs to targeting peptides or antibodies is usually accompanied by a decrease in the pharmacokinetic properties of the conjugate. One of the most common methods to couple two protein molecules together is to employ a heterobifunctional cross-linking agent whose functional moieties react with specific side chain groups in proteins (e.g. amine groups, sulfhydryl groups). Achieving specificity is a major challenge to this approach, however. For example, amine groups (e.g. lysine residues) occur in high frequency throughout protein sequences. Therefore, conjugation usually occurs randomly throughout the protein structure when amine-reactive agents are employed, often compromising the natural properties of these residues (e.g. ribosome inactivation). Conjugation to a specific residue provides the advantage of a predictable and reproducible structure. In this regard, use of cysteine side chains for targeting cross-linking reagents is advantageous. Cysteine residues are found at lower frequencies within protein sequences, and the conjugation reaction is more efficient using the highly reactive sulfhydryl group of the cysteine. Type 1 RIPs do not contain cysteine residues in their sequences. Saporin extracted from S. officinalis L. is one of the most commonly used type I RIPs for generating immunotoxins [23]. We have engineered a cysteine residue at position 255 of the sap-3 isomer with the intent of introducing a site for chemical conjugation. This makes the conjugation reaction more efficient and less complicated. Moreover, unlike other protein modification methods by
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which the modification could occur at any part of the saporin molecule, a single cysteine ensures a specific binding site. In our case, the important factors for the choice of the mutation site were: (1) the modification should not affect ribosome binding (i.e. should not be in the catalytic site of the saporin), (2) the engineered site should be easily accessible for conjugation and (3) the new protein should be stable. Previous studies show that all residues in the catalytic active site cleft of RIP proteins are conserved [15] and mutations at these catalytic active site cleft residues cause a drastic loss of activity [33]. In addition, our observations indicate that mutations in the N terminus produce unstable proteins (unpublished). One modification that meets each of these criteria is the introduction of a cysteine residue at position 255 of sap-3 isomer. The engineered recombinant saporin was expressed in BL(21) E. coli cells, where recombinant protein was expressed in the soluble fraction. Expression of the recombinant saporin gene We found that the Cys255sap-3 cloned into the pET11d plasmid achieves suitable expression in BL21 (DE3) pLysS cells, despite the protein’s toxicity. Following induction for protein expression, induced bacteria were lysed, sonicated and fractionated by centrifugation. Soluble and insoluble fractions were then analyzed by SDS–PAGE, followed by Western blot analysis using chicken anti-saporin antibody. The results revealed that Cys255sap-3 is expressed in soluble fraction, with no indication of Cys255sap-3 in the insoluble fraction (not shown). Expression yield was only slightly improved when induction time was extended to 4 h as compared to 2 h. Extraction and purification of the Cys255Sap-3 protein Recombinant sap-3 protein was then isolated using size-exclusion chromatography (Fig. 1). As may be seen, SDS–PAGE gels show a major band with an apparent MW of 30 kDa, slightly higher than sap-3 isomer, a 253 amino acid long protein. This difference in migration is likely due to the extra residues added to the recombinant saporin (260 amino acids, the last 7 amino acids added are, Ala, Cys, Glu, Lys, Asp, Glu and Leu). We also noted a minor impurity (at 26 kDa) that co-purifies with the recombinant saporin. Since this was not detected in the Western blot that utilizes a polyclonal antibody derived against the intact saporin protein, it is unlikely that this smaller band represents a proteolytic fragment of the saporin protein. The gels in Figs. 1–3 were all run under non-reducing conditions. With Coomassie Blue staining, no dimerization via a disulfide bridge involving Cys255 was evident. Thus, this protein provides a site with a low propensity to form intramolecular disulfide bonds, a useful property for sites that will later undergo covalent modification. However, using the more sensitive immunoblot method,
Fig. 1. SDS–PAG E (A) and Western blot (B) analysis of saporin, sap-3 isomer and Cys255sap-3. (A) SDS–PAGE analysis was done with nonreducing SDS gels. Lane 1: protein marker, lane 2: commercial saporin, lanes 3 and 4 Cys255sap-3, lane 5: sap-3 isomer. (B) Western blot assay using the polyclonal anti-saporin antibodies (dilution 0.36 lg/ml). Lane 1: protein marker, lane2: commercial saporin, lane 3: sap-3 isomer, lanes 4 and 5: Cys255sap-3.
a species at approximately twice the size of monomeric saporin was revealed. In summary, the purification described here provides a rapid reproducible and convenient method for obtaining yields of 2.7 mg/l culture (Table 1). The activity of the cysteine residue in the Cys255sap-3 To confirm the reactivity of Cys255 in the sap-3 protein, we first conjugated Cys255sap-3 with a fluorescent dye (Alexa 568 C5 maleimide, Invitrogen, CA), (Fig. 2, left panel) and then stained the same gel with coomassie (Fig. 2, right panel). Prior treatment of the Cys255sap-3 protein with the reducing agent Tris (2-carboxyethyl) phosphine Hydrochloride (TCEP HCl) before conjugating with fluorescent dye did not result in improved labeling levels. There were no visible bands for dimers under non-reducing SDS–PAGE gels (Figs. 1A and 3), but dimers were present in the Western blot (Fig. 1B). Such a difference is often encountered because of the sensitiv-
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Fig. 2. SDS–PAGE analysis of Alexa 568 tagged Cys255sap-3. Left column (lanes 1 and 2), image of non-reducing SDS–PAGE gel scanned on Typhoon Scanner using the 580 nm laser. Right column (lanes 3 and 4) Coomassie stained non-reducing SDS–PAGE gel (the same gel as left column). Lanes 1 and 4 protein marker, lanes 2 and 3: Alexa 568 tagged Cys255Sap-3, (2 lg).
ity differences between the two assays. Not detecting the bands in SDS gels indicates that the amount of dimerization is minimal. Thus, we concluded that Cys255 does not represent a position in the protein that has a high propensity to dimerize via disulfide bands. Such a feature would complicate the efficiency of modifying this site with cross-linking reagents. To validate that Cys255 in the recombinant saporin is suitable for cross-linking to antibody, we conjugated the Cys255sap-3 to a nonspecific mouse IgG. Coomassie stained SDS–PAGE showed that a high level of cross-linking was achieved when the heterobifunctional reagent was targeted to Cys255 in saporin (Fig. 3).
Fig. 3. SDS–PAGE analysis of recombinant saporin, mouse anti-human IgG and recombinant saporin conjugated to mouse anti-human IgG. Lane 1, protein marker, lane 2, Cys255sap-3 (2 lg), lane 3, mouse anti-human IgG (2.4 lg), lane 4, Cys255sap-3 conjugated to mouse anti-human IgG (3.8 lg). Table 1 Total protein and Cys255sap-3 contents at different stages of purification of recombinant Cys255sap-3 Fraction
Total protein amount, measured by BCA assay (per 1 L culture)
Total Cys255sap-3 amount (per 1 L culture)
Approximate yield (%)
Total cell lysate Soluble cell lysate After purification (95% purity)
350 mg 120 mg —
15.6 mg 12.5 mg 2.7 mg
100 80 17
The preparation was with 2.5 g wet weight cells from 1 L of culture.
Ribosome inactivating activity of the Cys255sap-3 Ribosome inactivating activity of Cys255sap-3 expressed in BL21 (DE3) pLysS cells was tested by in vitro translation inhibition assay using rabbit reticulocyte lysates. The enzymatic activity of recombinant Cys255sap-3 was compared to those of sap-3 isomer and commercial saporin purchased from Advanced Targeting Systems. Fig. 4, a semi-log graph, shows the protein synthesis (% control) as a function of concentration where
concentration is expressed in log values. These values (10 12.0744 for Cys255sap-3, 10 11.2978 for sap-3 isomer and 10 11.5014 for commercial saporin) were calculated by MatLab using the experimental data (three replicates at each point) as seen in the graph. IC50 values were than calculated by taking anti-log values of concentration values. The purified Cys255sap-3 displayed a strong inhibitory activity on protein synthesis in the cell-free rabbit
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Fig. 4. Semi-log graph showing the protein synthesis inhibition as a function of toxin concentration. Activities for Cys255sap-3, Sap-3 isomer and commercial saporin were tested by protein synthesis inhibition assay. One microliter of mRNA was added to 0.5 ml RNase-free microcentrifuge tubes and incubated 3 min at 65 C. One microliter of samples (dilutions of Cys255sap-3, Sap-3 isomer and commercial saporin), 7 ll of rabbit reticulocyte lysate (Promega), 1 ll of amino acid minus mixture and 0.2 ll of RNasin were added to respective tubes and incubated at 30 C in a water bath for 30 min. Two microliters of samples from each tube were added on 96-well opaque plate and read on L-Max luminometer using 25 ll Luciferase Assay Reagent (Promega). Data were analyzed using MatLab. Green, Sap-3 isomer; Blue, Cys255sap-3; Red, commercial saporin (purchased from Advanced Targeting Systems).
reticulocyte system, with an IC50 of 0.84 · 10 12. This value is on the order of native sap-3 isomer (5.0 · 10 12) and commercial saporin (3.1 · 10 12) (Fig. 4). In conclusion, Cys255 is a good position to target/place the thiol group because it does not have a high propensity to form intermolecular disulfides, it provides good linker flexibility, and modification at this site does not reduce saporin activity.
[5]
[6]
[7] [8]
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