1SibTech, Inc., Newington, CT 06111, USA and 2Rammelkamp Center for Research, Case Western Reserve University School of Medicine, Cleveland, OH 44109, USA
3 To whom correspondence should be addressed. e-mail: mbacker{at}sibtech.com
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Abstract |
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Keywords: adapter protein/chimeric RNase/delivery complexes/targeting drugs
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Introduction |
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We have recently proposed a novel strategy for modular assembly of targeting complexes for drug delivery (Backer et al., 2002a). This technology avoids chemical modification of targeting proteins by using a standardized docking system that includes two modules: a docking tag fused to a targeting protein and a payload module containing an adapter protein for binding to the docking tag. Standardized payload modules are pre-made by linking a cargo to an adapter (Figure 1).
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This is a somewhat cumbersome and expensive strategy for preparing the quantities of HuS that are necessary for pre-clinical and early clinical studies. Here we describe a strategy for making native 21127 amino acid HuS using an engineered chimeric bovine/human RNase (BH-RNase) as a starting material. This strategy is based on a procedure developed for preparation of S-protein via subtilisin cleavage of RNase A between A20 and S21 (Richards and Vithayathil, 1959). Unfortunately, limited proteolytic cleavage of RNase I does not release HuS, presumably because of incompatibility of the hinge loop 1525 with the subtilisin active center (Gupta et al., 1999
; Pous et al., 2001
). To reconstruct a cleavage site in this loop, we have constructed BH-RNase containing 129 amino acids of RNase A and 30127 amino acids of RNase I. The 2129 region of RNase A differs from a corresponding region of RNase I by the presence of N24 instead of T24. In order to reconstruct wild-type RNase I sequence starting from S21, we have also constructed a mutant BH-RNase (N24T). We found that both chimeric RNases can be expressed at a high level, refolded into an enzymatically active conformation, and cleaved by either subtilisin or proteinase K between A20 and S21. The affinity of HuS obtained from BH-RNase to Hu-tagged proteins is
5-fold higher than that of recombinant 18127HuS(P19A, S20A), reflecting a higher proportion of functionally active protein.
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Materials and methods |
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The pET-HP plasmid encoding 1127 amino acids of human RNase I (el-Joubary et al., 1999) was a generous gift from Dr G.DAlessio (Napoli Federico II University, Naples, Italy). The ORF of an 18125 fragment of human RNase I with the T24N amino acid substitution was amplified by PCR using a 5'-ATGTCCGCTGCCAGCAGCTCCAACTACTGCAACCAGATGATGCGTCG-3' sense primer (mutation underlined) and an antisense primer, 5'-CTATTCAACACACGCGTCGAA ATGAACCGG-3'. The PCR product was cloned in the pETBlue-1 using a Perfectly Blunt Cloning Kit (Novagen), and the T24N mutation was confirmed by sequencing. Then, 10 bovine RNase A codons were introduced by PCR using a sense primer, 5'-TTTGAGCGGCAGCACATGGACTCCAGCAC TTCCGCTGCCAGCAGCTCC-3'. An antisense primer, 5'-TCAAGAGTCTTCAACAGACGCGTCG-3', introduced codons for D126, S127 and a stop-codon. The PCR product was cloned in the pETBlue-1 and the resulting construct was confirmed by sequencing. Seven more bovine codons were introduced in the sequence by amplifying it with a sense primer, 5'-ATGAAGGAAACTGCAGCAGCCAAGTTT GAGCGGCAGCACATGGACTCC-3' and the above antisense primer. The PCR product was cloned in the pETBlue-1 and confirmed by sequencing. The 129B/30127H-RNase coding sequence was amplified by PCR using a sense primer, 5'-CACAAGCATATGAAGGAAACTGCAGCAGCC AAG-3' (NdeI site underlined) and an antisense primer, 5'-TACGGTACCTCAAGAGTCTTCAACAGACGCGTCG-3' (KpnI site underlined), and cloned in NdeIKpnI sites of the pET29a(+) bacterial expression vector (Novagen). The N24T amino acid substitution was introduced in the pET29/129B/30127H-RNase plasmid DNA by site-directed mutagenesis using a Gene-Tailor Site Directed Mutagenesis Kit (Invitrogen), a sense primer, 5'-TCCGCTGCCAGCAGC TCCACCTACTGCAACCAG-3' (mutation underlined) and an antisense primer, 5'-GGAGCTGCTGGCAGCGGA AGTGCTGGAG-3'. The N24T mutation was confirmed by sequencing.
Purification of HuS
Both 129B/30127H-RNase (BH-RNase) and 129B/30127H-N24T-RNase (BH-N24T) were expressed in BL21(DE3) E.coli (Novagen), refolded from inclusion bodies, and purified by ion-exchange chromatography on a HiTrap SP-Fast Flow column (Amersham) and RP-HPLC on C8 as described for recombinant RNase I (Backer et al., 2003). To exchange buffer, RP-HPLC purified proteins were loaded on a HiTrap SP-Fast Flow column equilibrated with a buffer containing 20 mM NaOAc pH 6.5 and eluted with a buffer containing 20 mM NaOAc, 0.6 M NaCl, pH 6.5. BH-RNase and its mutant were digested with either subtilisin or proteinase K (Sigma) at a protein to protease ratio of 100:1 (w/w) at room temperature or at 4°C. Bovine peptide was removed from the digestion mixture by RP-HPLC on a C18 column as described (Backer et al., 2002b
, 2003). After buffer exchange on a HiTrap SP-Fast Flow column performed as described above, HuS-T24N and wild-type HuS were purified by affinity chromatography on a Hu-peptide column. The column was prepared by coupling of a CA-extended Hu-peptide (CA-KESRAKKFQRQHMDS synthesized by Genemed Synthesis, Inc., South San Francisco, CA) to Activated Thiol Sepharose 4B (Amersham) according to the manufacturers protocol. Before coupling, CA-Hu-peptide was treated with 100 mM TCEP for 30 min at room temperature, purified on an RP-HPLC C18 column, and transferred into conjugation buffer via buffer exchange on a HiTrap SP-Fast Flow column. Bound HuS was eluted from the affinity column with 0.2 M citric acid. After buffer exchange on a HiTrap SP-Fast Flow column (as described above), proteins were aliquoted and stored at 70°C.
HuS/Hu-tag binding assay
The equilibrium dissociation constant (KD) values for HuS-T24N/Hupeptide complexes were determined as described (Backer, 2002b, 2003). Briefly, HuS-T24N at final concentrations of 0.1, 0.2 or 0.3 nM was mixed with varying amounts of Hu-tagged vascular endothelial growth factor [Hu-VEGF, expressed as described in Backer et al. (Backer et al., 2002a
)] in a buffer containing 20 mM TrisHCl, 100 mM NaCl, pH 7.5, 0.1 mg/ml poly C and incubated for 5 min at room temperature. The activities of the reconstituted ribonucleases were measured by the absorbance at 280 nm of the reaction mixtures cleared from TCA-precipitated material. One optical unit of TCA-soluble material released from poly C incubated with reconstituted ribonuclease was defined as one relative unit of ribonuclease activity. Calculations of the KD values were performed assuming that the initial rate of the hydrolysis is proportional to the concentration of reconstituted ribonuclease. DYNAFIT software was used for the global fitting with numeric iteration and calculation of the KD values (Kuzmic, 1996
).
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Results |
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Bovine RNase A is cleaved by subtilisin between A20 and S21 yielding functionally active S-protein (Richards and Vithayathil, 1959). In contrast, limited proteolytic cleavage of human RNase I does not release HuS, presumably because of incompatibility of the hinge loop 1525 with the subtilisin active center, most likely due to a conformational change induced by P19 (Gupta et al., 1999
; Pous et al., 2001
). Indeed, direct comparison of the bovine and human hinge loops 1525 backbones (Figure 2A, shown in green and red respectively) revealed significant differences between the two RNases.
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Both BH-RNase and BH-N24T were expressed in E.coli strain BL21(DE3), recovered from inclusion bodies, refolded by a two-step dialysis, and purified via ion-exchange and reverse-phase chromatography as described previously for recombinant RNase I (Backer et al., 2003). Purified BH-RNase and BH-N24T had typical yields of 6080 mg/l, and were found to be >98% pure judging by SDSPAGE (Figure 2D, for BH-RNase) and HPLC (data not shown). Ribonuclease activities of BH-RNase and BH-N24T were indistinguishable, both were
2-fold lower than that of recombinant RNase I (prepared as described in Backer et al., 2003
), and varied from isolation to isolation within 20% (data not shown).
BH-RNase cleavage
Initially, limited proteolytic digestion of BH-RNase with subtilisin was performed at the BH-RNase/protease ratio of 100:1 (w/w) at room temperature. A BH-RNase fragment with an electrophoretic mobility corresponding to that of HuS was transiently accumulated within 1 h under these conditions (Figure 3A, top). However, significant quantities of BH-RNase were degraded by 3060 min of incubation. Lowering the incubation temperature to 4°C significantly reduced the reaction rate and enhanced the selective proteolysis of the A20S21 peptide bond, yielding more HuS(T24N) (Figure 3A, bottom).
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HuS purification
Short peptides released from BH-RNase and BH-N24T during proteolysis were removed from digestion mixtures by RP-HPLC on a C8 column as described for separation of human S-peptide and recombinant 18127HuS(P19A, S20A) (Backer et al., 2003). In order to separate HuS fragments from undigested ribonucleases we used an affinity column that was constructed by coupling of a CA-extended Hu-peptide (CA-KESRAKKFQRQHMDS). After passing reaction mixtures through this column, undigested RNase and minor fragments were found in flow-through fractions, whereas HuS fragments bound to the column were eluted with 0.2 M citric acid (Figure 4, for BH-RNase cleavage). Q-TOF ES MS confirmed the identity of HuS(T24N) (MW 12 146) as a main product obtained from BH-RNase. In addition, 510% of purified protein resulted from the cleavage at the S21S22 bond, as was reported for RNase A (Neumann and Hofsteenge, 1994
; Mendez et al., 2000
).
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To characterize the affinity of purified HuS(T24N) to Hu-peptide, we used VEGF fused to Hu-tag [Hu-VEGF, expressed as described in Backer et al. (Backer et al., 2003)]. Recon stituted ribonuclease activity in HuS/Hu-VEGF mixtures was measured under conditions of equilibrium between free and bound HuS(T24N) as described (Backer et al., 2002b
, 2003). To derive equilibrium dissociation constant (KD) values for these complexes, the experimental data were analyzed with DYNAFIT software for the global fitting with numeric iteration and calculation of the KD values (Kuzmic, 1996
) under an assumption that the initial rate of the hydrolysis is proportional to the concentration of reconstituted ribonuclease. We found that the affinity of HuS(T24N) to Hu-VEGF was characterized by a KD value of 29.3 ± 2.6 nM (Figure 5). These values are approximately five times lower that that obtained for complexes between recombinant 18127HuS(P19A, S20A) and Hu-VEGF under the same experimental conditions (KD of 162 ± 16 nM; Backer et al., 2003
). To explain this difference, we have tested what proportion of 18127HuS(P19A, S20A) binds to the Hu-peptide affinity column and found that only 2030% of this protein was retained on the column (data not shown). The latter result supports the notion that the difference in KD values is due to significant heterogeneity of refolded recombinant 18127HuS(P19A, S20A) as compared with HuS preparations.
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Discussion |
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We found that BH-RNase and its N24T mutant are cleaved by both subtilisin and proteinase K at the A20S21 bond, yielding functionally active HuS fragments. The conditions of cleavage and affinity purification on the S-peptide column are optimized to achieve a 4050% yield of HuS fragments from chimeric RNases (Figures 3 and 4). Importantly, HuS displays an 5-fold higher affinity to Hu-tagged proteins than 18127HuS(P19A, S20A) [Figure 5 and Backer et al. (Backer et al., 2003
)]. This change might be a result of more homogeneous preparations of HuS that arise upon proteolytic cleavage of chimeric RNase. Indeed, surface plasmon resonance experiments and affinity chromatography on the S-peptide column suggest that refolded 18127HuS(P19A, S20A) exists in several conformations with different affinities to Hu-peptide (Backer et al., 2003
).
We found that the BH-N24T mutant appeared to be more resistant to proteolytic cleavage by subtilisin and proteinase K (Figure 3). This result is somewhat surprising, since molecular modeling indicates that an amino acid in position 24 is not involved in direct interactions with a catalytic triad in subtilisin or proteinase K (data not shown). Unfortunately, this resistance did not lead to a higher yield of wild-type HuS. Nevertheless, since wild-type HuS might be more advantageous in minimizing the risk of an adverse immunological response in potential clinical applications of the adapter/docking tag system, experiments to optimize its production are in progress.
Taken together, our data indicate that chimeric BH-RNase is a superior source of an adapter protein for assembled complexes for targeted drug delivery. Our recent experiments with tumor radionuclide imaging using techetium-99m labeled HuS/Hu-VEGF complexes suggest important new applica tions for this system (F.Blankenberg et al., manuscript in preparation).
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Received June 9, 2003; revised August 4, 2003; accepted August 28, 2003.