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Antimicrobial Agents and Chemotherapy, September 2001, p. 2517-2523, Vol. 45, No. 9
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.9.2517-2523.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Isolation of Rat Dihydrofolate Reductase Gene and Characterization of Recombinant Enzyme

Structural Biology Department, Hauptman Woodward Medical Research Institute, Buffalo, New York 14203,¹ and Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202²

Received 11 December 2000/Returned for modification 5 March 2001/Accepted 18 June 2001

	ABSTRACT

Top Abstract Introduction Materials and Methods Results and Discussion References

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, which serves as a substrate for a number of one-carbon transfer reactions in purine and pyrimidine synthesis, including that of thymidylate. DHFR, along with other enzymes in the folate metabolic pathway, is critical for the biosynthesis of DNA, RNA, and certain amino acids (2-4, 51). Consequently, inhibition of DHFR enzymatic activity depletes the tetrahydrofolate pool inside the cell and inhibits DNA synthesis, subsequently leading to cell death. For this reason, DHFR has been studied extensively and many antifolates have been synthesized and tested as potential candidates for drugs (5, 19-24, 28, 30, 35, 44-48, 52). More recently, antifolates have been shown effective against such opportunistic infectious agents as Pneumocystis carinii and Toxoplasma gondii (1, 6, 9, 10). Since immunosuppressed patients and those with AIDS are severly affected by these pathogens, efforts have been focused on the design of antifolates that are selective against P. carinii DHFR (pc DHFR) and T. gondii (or tgDHFR) (12, 18, 22-24, 27, 28, 33, 42, 44, 48). In most of these studies, antifolate selectivity reported as a ratio of 50% inhibitory concentrations (IC₅₀s) from two DHFR species was measured against crude DHFR preparations from rat liver (44, 47, 48).

Evidence from sequence analysis and three-dimensional crystal structures of DHFR from many species shows that there is a high degree of conservation at the primary sequence and structural level among DHFRs (8, 10-14, 17, 19, 29, 34, 36, 37, 41, 43, 50, 53, 55). However, kinetic and biochemical characterization data reveal differences in the mechanism of action that result in significant species specificity by selected inhibitors (15-17, 21, 25, 26, 31, 36, 39, 43). For example, trimethoprim (TMP) [2,4-diamino-5-(3,4,5,-trimethoxybenzyl)-pyrimidine], which is used against bacterial infections, demonstrates a significantly higher affinity for bacterial DHFR than for eukaryotic DHFR. While a drug such as TMP also shows reasonable selectivity against pc- and tgDHFR, it is not a particularly effective inhibitor, as indicated by its high IC₅₀s (2-4, 51). Clearly, drugs with high potency as well as selectivity for effective therapy are still in demand.

Despite the availability of crystal structures of DHFR from many species and the extensive body of literature on the effects of compounds against rat liver DHFR, neither the DNA sequence nor the protein structure of the rat enzyme is known. Because of a high degree of similarity in the N-terminal sequence of rat and human DHFR (38, 54), it was assumed that the rat enzyme would be a faithful representation of the human enzyme. However, only a detailed comparison of its sequence, structure, and kinetics with the human enzyme will provide the needed data. Therefore, we have isolated the DHFR gene from a rat cDNA library and expressed an active form of recombinant DHFR protein from Escherichia coli. Inhibitory data for select antifolates reveals the expected pattern of homologies; however, one example shows significant differences in the rat and human DHFR IC₅₀s.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Reagents. A rat (6-month-old Sprague Dawley male) liver lambda cDNA library for isolation of rat liver DHFR cDNA was purchased from Stratagene (Cedar Creek, Tex.). Gateway plasmids pDEST-15 and pDONR201 for the construction of expression clones as well as the required clonase enzymes were obtained from Life Technology (Bethesda, Md.). The pCRII vector and the TA cloning kit for the cloning of PCR products were from Invitrogen (Carlsbad, Calif.). Different E. coli strains were used for DNA manipulations and for protein expression: DH5 [supE44 lacU169 (80 lacZM15) hsdR71 recA1 endA1 gyrA96 thi-1 relA1] and DB3.1 [F gyrA462 endA (Sr1-recA) mcrB mrr hsdS20(r_B m_B) supE44 ara-14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 leu mtl1] were from Life Technology, and BL21(DE3) [hsdS gal (cIts857 ind1 S am7 nin5 lacUV5-T7 gene 1] were from Novagen (Madison, Wis.). Restriction endonucleases and T4 DNA ligase were from New England Biolabs (Beverly, Mass.), Taq DNA polymerase was from Sigma (St. Louis, Mo.) and Life Technology, and pCRII vector was from Stratagene. Radioactive isotope [-³²P]dATP used for Southern hybridization was obtained from Pharmacia-Amersham (Piscataway, N.J.). The Random Priming DNA Synthesis kit for making radioactive DNA probes was purchased from Stratagene. Nylon dot blot membranes were obtained from Osmonics (Minnetonka, Mont.). Positive cDNA lambda clones were visualized using a PhosphorImager scanner (Pharmacia-Amersham). All other chemicals and reagents were obtained from Sigma. All chromatography columns were from Pharmacia-Amersham, and the BIOCAD 700E Perfusion Chromatography workstation was from PE-Biosystems (Norwalk, Conn.).

Library screening. A mouse DHFR cDNA fragment was initially generated from mouse liver total mRNA through reverse transcription-PCR. First-strand cDNA was synthesized with a reverse primer complementary to a 3'-untranslated region (UTR) of mouse DHFR: 5'-CGG GAT CCC CTC TCT AAA GAA AGA ATA ACT CAT AGA TCT AAA GCC-3'. It was subsequently amplified with a PCR primer pair, 5'-CGG GAT CCA TGG TTC GAC CGC TGA ACT GCA TCG-3' and 5'-CGG GAT CCA AGT CCC ATG GTC TTG TAA AAA TGC-3'. The resulting 677-bp fragment containing the full-length mouse DHFR open reading frame (ORF) was cloned into a pCRII vector (Invitrogen) using the TA cloning kit and sequenced to confirm the identity of the mouse DHFR sequence. This 677-bp fragment was then used as a probe to screen a rat liver cDNA lambda library (Stratagene) with a titer of ~1.75 × 10⁷ PFU/µl. Approximately 1,000,000 plaques of the library were screened using 20 150-mm-diameter NZY agar plates. Briefly, 20 sets of 600 µl of freshly grown E. coli XL1-Blue MRF' [(mcrA) 183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F' proAB lac1^q ZM15 Tn10 (Tet)] cells at an optical density at 600 nm (OD₆₀₀) of 0.5 per plate were each infected with 1 × 10⁵ plaques from cDNA library and incubated at 37°C for about 8 h. Plaque lifts were made in duplicate immediately and lysed in situ. Phage DNA was denatured and fixed on the membrane through baking in a vacuum oven for 2 h at 80°C. Membranes were then hybridized to a ³²P-radiolabeled probe from a mouse DHFR PCR fragment to detect cross-reacting plaques by using the random priming kit from Stratagene (49). Positive plaques were diluted and repeatedly hybridized in duplicate until individual homogeneous clones could be isolated. After obtaining the positive plaque stocks, clones were incubated with the ExAssist helper phage and then introduced into a nonsuppressing E. coli SOLR {e14 (McrA) (mcrCB-hsdSMR-mrr) 171 sbcC recB recJ uvrC umuC::Tn5(Kan^r) lac gyrA96 relA1 thi-1 endA1^R [F' proAB lac1^q ZM15] Su} strain for the excision of pBlueScript phagemid containing the positive cDNA insert.

DNA sequence analysis. Multiple positive pBlueScript clones containing rat liver DHFR cDNA inserts were sequenced using an ABI PRISM automated sequencer from Roswell Park Cancer Institute Biopolymer Facility (Buffalo, N.Y.). T7 forward and M13 reverse primers on the vector were used to sequence and confirm the isolation of the full-length rat liver DHFR cDNA. The rat liver DHFR cDNA sequence was compared with known DHFR sequences from different species using BLAST web services from the National Center of Biotechnology Information (NCBI). Protein sequence alignment with other known DHFR species was performed using the Wisconsin Package Version 10.0 (TRANSLATE, PILEUP, REFORMAT), Genetics Computer Group, Madison, Wis.

Heterologous expression of rat liver DHFR gene. The Gateway cloning kit (Life Technology) was used to construct expression vectors for the rat liver DHFR gene. The rat DHFR gene was PCR amplified with a pair of oligonucleotide primers harboring the attB bacteriophage recombination sites as well as the tobacco etch virus (TEV) protease recognition site (ENLYQG). The PCR product was introduced into the entry vector containing the attP bacteriophage recombination site via the attB × attP recombination reaction. The resulting entry vector now contained an attL recombination site that could be recombined into the destination vector with the matching attR site. The reactions, designated BP and LR reactions, respectively, were performed according to the manufacturer's recommended conditions (Life Technology). The resulting expression clone, pDest15-rDHFR, would express a glutathione S-transferase rat DHFR (GST-rDHFR) fusion protein in bacteria and would contain a TEV protease recognition site between the GST and rat DHFR domains. The forward primer contains the attB1 recombination site and the TEV protease recognition site fused in frame with the N-terminal sequences of rat DHFR (5'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AGA AAA TCT GTA CTT CCA GGG GAT GGT TCG TCC GCT GAA CTG CAT CGT CGC C-3'). The reverse primer contained the attB2 recombination site fused with a complementary region of the 3'-UTR of the rat DHFR (5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG GAT CCA GCA GAA GTG GTC TTA TAA AAT GC-3').

The pDEST15-rDHFR plasmid was sequenced to verify the presence and integrity of the rat DHFR insert. The plasmid was subsequently introduced into the BL21(DE3) E. coli strain via chemical transformation as previously described (49). Cells containing the plasmids were grown at 30°C to an OD₆₀₀ of 0.4 and were induced with 0.15 mM isopropyl--D-thio-galactopyranoside (IPTG) for 4 h at 30°C. Expression of the GST-rDHFR fusion protein was confirmed through the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell lysate after the IPTG induction. The typical yield of soluble GST-rDHFR fusion protein was between 35 and 40 mg per liter of cell culture after purification.

Purification of GST-rDHFR and rat DHFR proteins. To purify the GST-rDHFR fusion protein, a GST affinity column was used. After induction with IPTG, cells were harvested, resuspended in lysis buffer (50 mM KH₂PO₄ [pH 7.0] 50 mM KCl, and 1 mM EDTA), and lysed with lysozyme (50 mg for 4 liters of cell culture) in the presence of protease inhibitors. To optimize lysis, cell suspension also went through a liquid nitrogen freeze-thaw cycle twice. Lysed cells were immediately centrifuged at 40,000 rpm for 45 min at 4°C (45 Ti rotor, L8 Beckman ultracentrifuge). The clear cell lysate was filtered through a 0.45-µm-pore-size membrane and applied to the GST affinity column on a BioCAD 700E perfusion chromatography workstation. Bound GST-rDHFR fusion protein was eluted with elution buffer (50 mM Tris-HCl [pH 8.0], 25 mM glutathione). The eluted fractions were then digested with TEV protease at 30°C overnight in TEV digestion buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 5 mM DTT). After digestion with TEV, one glycine residue from the TEV recognition site should have been left at the N terminus of the rat DHFR protein. The digestion mixture was loaded onto a G-25 desalt column to exchange the buffer composition with that of the lysis buffer but at pH 8.0. Rat DHFR protein was separated from GST and undigested GST-rDHFR fusion by passage through a second GST affinity column. The rat DHFR protein was collected as the flowthrough fraction. A final G-75 gel filtration column removed any other potential impurities of different sizes.

Enzyme and inhibitor assay. The activities of both the recombinant rat liver DHFR and the GST-rDHFR fusion protein were measured at room temperature spectrophotometrically at 340 nm for a decrease in absorbance which occurs when NADPH and dihydrofolate are converted to NADP⁺ and tetrahydrofolate. The assay was performed essentially as described by Prendergast et al. (43).

The spectrophotometric assay (micromolar IC₅₀ measurement) used to measure the abilities of compounds to decrease the rate of enzymatic reduction of dihydrofolate to tetrahydrofolate in the presence of NADPH was performed at 37°C with saturating concentrations of substrate and cofactor, and with 150 mM KCl, as previously described (6, 9, 44).

GenBank nucleotide sequence accession number. The GenBank accession number for full-length rat liver DHFR cDNA is AF318150.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Isolation of rat DHFR gene. The rat liver DHFR gene was isolated through the screening of a rat liver cDNA library by using a mouse DHFR probe. After multiple rounds of screening, one clone was obtained that contained a full-length ORF. The nucleotide sequence of the gene plus portions of the 5'- and 3'-UTRs are shown in Fig. 1.

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Shown is the DNA sequence and predicted amino acids sequence of the rat DHFR gene, with portions of the 5' and 3' flanking regions. The translation of the gene starts at position 88 and ends at nucleotide 648. The polypeptide sequence in single-letter coding is indicated below the DNA sequence. The GenBank accession number for the rat DHFR gene is AF318150.

The 561-bp ORF encodes a protein of 187 amino acids. Alignment of the putative translated product of this gene with other mammalian DHFR proteins revealed significant homology, with 96% primary sequence identity with mouse and Chinese hamster DHFR and 89% with human DHFR (Fig. 2). Comparison of DHFR proteins from opportunistic fungi revealed an identity of 41% to human-P. carinii and rat-P. carinii DHFR and 31% to a protozoal parasite T. gondii DHFR. The active enzymatic sites between rat and other DHFR proteins demonstrate a higher level of conservation. Thirty-three out of 35 amino acids within the primary sequence that constitute the active enzymatic site, as well as the sites for substrate and cofactor binding, are identical between human and rat. Thirty-four out of 35 are identical between rat and mouse and Chinese hamster (Fig. 2). Eleven amino acids (Fig. 2) that are conserved among all known DHFR proteins are also preserved in the rat protein isolated, which aligns perfectly with other known DHFRs.

View larger version (64K): [in this window] [in a new window]   FIG. 2.   Protein sequence comparisons of rat DHFR with six other known DHFR enzymes. Highlighted amino acid residues represent conserved regions among different species. Light gray boxes indicate identity among the seven DHFR proteins, and dark gray boxes represents significantly similar residues.  and *, residues that surround the active site of the DHFR enzyme; some residues (*) are conserved in all known DHFR. Sequence alignment was performed using the Wisconsin Package version 10.0 (TRANSLATE, PILEUP, REFORMAT), Genetic Computer Group.

Comparison of rat liver DHFR protein with other known DHFRs indicated a high level of conservation for DHFR proteins among higher eukaryotes. Functionally important residues within the DHFR enzyme demonstrate an even higher level of conservation. The Phe³⁴, Phe³¹, Leu²², Ile⁷, Ile⁶⁴, Leu⁶⁷, and Glu³⁰ residues that are important for dihydrofolate and other ligand binding (2) are conserved in rat DHFR. Lys⁵⁴, which has been shown to be important for the association of cofactors NADPH and NADP (26), are the same in rat DHFR. Overall, the organization of rat DHFR implies a structure similar to those known for other DHFR enzymes. X-ray crystallographic studies are underway to validate the structural homology of the rat DHFR structure with that of human DHFR.

Expression and purification of recombinant rat DHFR protein. The putative rat DHFR gene was introduced into a Gateway expression plasmid via two steps of recombination reactions. The resulting expression clone produces a GST fusion protein in BL21(DE3) cells upon induction by IPTG. The recombinant GST fusion protein, tentatively named GST-rDHFR, was expressed as a 50.2-kDa protein with the GST and rat DHFR moiety linked by a TEV protease recognition site. As shown in Fig. 3, the target GST fusion protein was separated from total cellular proteins by the affinity columns (lanes 1 and 2). A minor band with a molecular mass of approximately 29 kDa below the dominant GST fusion band in lane 2 is a truncated translational product of the GST portion and can be removed by a second round of GST affinity chromatography. Since no TEV protease recognition site is present within the putative rat DHFR protein, it could be separated from the GST portion after the digestion of TEV protease (Fig. 3, lane 3). Digestion yielded the expected 21.6-kDa putative rat DHFR protein and the 28.7-kDa GST protein. Subsequently, target rat protein was purified through additional rounds of GST affinity chromatography to remove the GST tags. After the final chromatographic purification using a G-75 sizing column, a single band on the SDS-PAGE gel (Fig. 3, lane 6) indicated the successful isolation of a highly pure and homogeneous protein.

View larger version (68K): [in this window] [in a new window]   FIG. 3.   Expression and purification of recombinant rat DHFR enzyme in BL21(DE3) cells. Proteins were assayed on SDS-12% PAGE gels. Lane 1, molecular mass markers, with sizes of standard fragments (in kilodaltons) indicated on the left; lane 2, cell lysate with overexpressed GST-rDHFR fusion; lane 3, peak fraction after the GST affinity chromatography: lanes 4 and 5, peak fraction of GST column digested with TEV protease; lane 6, purified rat DHFR recombinant protein after the G-75 gel filtration column. The sizes of the GST fusion protein, GST alone, and the pure rat DHFR protein are on the right.

All fractions during the purification process were assayed for the DHFR enzymatic activity that reduces dihydrofolate to tetrahydrofolate in the presence of NADPH. Monitoring the decrease of absorbance at 340 nm spectrophotometrically due to the conversion of dihydrofolate to tetrahydrofolate revealed that both the GST fusion protein and the pure rat moiety released from the fusion demonstrate high levels of DHFR enzymatic activity. However, the pure rat protein without the GST tag is about 30-fold more catalytically active than the GST fusion (data not shown). Our results indicate that we have isolated the DHFR gene from rat liver and have successfully expressed an active form of DHFR recombinant protein.

Inhibition of DHFR enzymatic activity with different compounds. Based upon similarities in N-terminal analysis of the native rat and human proteins (38, 54), many inhibitor binding assays of DHFR have been performed with rat liver enzyme (44, 46). We tested the inhibition of recombinant rat liver DHFR by several compounds and compared these results with those obtained from the native DHFR preparations (Table 1).

View this table: [in this window] [in a new window]   TABLE 1.   Comparison of micromolar IC50s of seven known inhibitors against various forms of rat and human DHFR and the selectivity ratio of those inhibitors against pcDHFRa

Seven antifolates which reflect a broad spectrum of inhibition and structural features were used to compare potential differences between inhibition of the recombinant and native rat liver DHFR enzyme (Table 1). Methotrexate (MTX) and MTXO represent classical pteridine analogues. PT652 and PT653 belong to a diaminopteridine class with a bulky planar tricyclic moiety, which were recently synthesized and tested for their potential potency and selectivity against pcDHFR (46), and trimethoprim, pyrimethamine, and TAB are lipophilic 2,4-diaminopyrimidine analogues.

A parallel inhibitory pattern of the compounds against the recombinant rat liver DHFR and the GST-rDHFR fusion protein was observed (Table 1). The GST-rDHFR fusion protein can catalyze the reduction of dihydrofolate, but with less efficiency than DHFR without the GST amino terminus. The turnover value (moles of product formed per minute per mole of enzyme) for the GST-rDHFR fusion is 300,000/min, whereas the value for the unfused DHFR is 10,000,000/min. Despite the reduced enzymatic activity, the inhibition profile of compounds with different potency was clearly reproduced with the GST fusion, indicating that the active site in the fusion protein is accessible to those inhibitors and that these inhibitors associate with the enzyme with similar kinetics.

When recombinant rat DHFR protein was used to test these inhibitors the potencies of most compounds showed a similar pattern to the IC₅₀s previously reported for rat (Table 1). The difference between the IC₅₀s of recombinant and native enzymes does not exceed threefold. The compounds that demonstrated the largest differences are PT652 and PT653 (Table 1).

A recombinant human DHFR enzyme was also used to determine the comparative efficiency of inhibitors against the rat and human enzymes. As shown in Table 1, the broad spectrum of potency in rat was reproduced with human recombinant DHFR. The largest difference between human and rat recombinant DHFR IC₅₀s is fivefold for compound PT652. The sensitivity of the human enzyme to PT652 is approximately ninefold less compared to that of the native rat enzyme (Table 1). In the case of PT653, the IC₅₀s for recombinant rat and human DHFR are comparable. However, PT653 is more sensitive for recombinant rat enzyme whereas PT652 is more sensitive for the native protein. Other than PT652 and PT653, the selectivity ratios between recombinant rat-to-human DHFR and native rat-to-human DHFR indicated a consistency between the recombinant and the native enzyme. All compounds tested, however, demonstrated lower IC₅₀s against recombinant rat DHFR than the human counterpart (Table 1).

For comparison, the inhibitory profiles of the seven compounds against pcDHFR were also included and the selectivity ratios against different forms of rat and human DHFR are shown (Table 1, last three columns). Again, most compounds demonstrate a similar selectivity ratio against pcDHFR for either the native or the recombinant rat DHFR. PT652 and PT653 had different sensitivities for the rat enzyme, and the largest variation was obtained with PT653, where the selection ratio was approximately 14.3-fold greater against pcDHFR for the native rat enzyme but was reduced to about 6.7-fold greater for recombinant enzyme. For PT652, there was no selectivity against pcDHFR with either the native or the recombinant rat DHFR. However, for the human enzyme, a moderate selectivity was observed (44). For the rest of the compounds, selectivity against pcDHFR appears to be consistent with all three forms of mammalian DHFR, with the selectivity ratio of the human enzyme being higher than that of the rat enzyme.

Summary. Rat liver DHFR has been used as the in vivo model to study hundreds of antifolates and related compounds against human DHFR for their potential drug implications, despite the fact that the sequence of neither the gene nor the protein was known. To better correlate the published literature for rat liver DHFR and human DHFR, we have cloned and sequenced the DHFR gene from a rat liver cDNA library. With a GST fusion tag, we expressed and purified an active form of recombinant rat DHFR protein in large quantity in E. coli cells.

Seven inhibitors chosen to represent different categories of antifolates with various selectivity against DHFR from the fungal pathogen P. carinii were tested against the recombinant rat liver enzyme. Similar inhibition profiles against both the rat and human enzyme were observed despite the fact that the IC₅₀s of recombinant rat DHFR were consistently lower than those of the recombinant human protein. The inhibitors also demonstrate consistent pcDHFR selectivity with both human and rat DHFR despite some variations with certain compounds. As noted, compounds with large bulky hydrophobic groups as found in PT652 and PT653 showed inconsistencies among the various assays. Conceivably, while binding of these antifolates provide a reasonable portrayal of their interactions with human DHFR, structural variations between human and rat enzyme coupled with unique features in the inhibitors could lead to differences in enzyme sensitivity and selectivity.

	ACKNOWLEDGMENTS

We thank Carol Yarborough for the assistance on the chromatographic work.

This work was supported in part by funds from GM51670 (V.C.), the Greater Buffalo Community Fund (V.C.), and the Wendt Foundation (J.A.B.).

	FOOTNOTES

^* Corresponding author. Mailing address: Structural Biology Department, Hauptman Woodward Medical Research Institute, 73 High St., Buffalo, NY 14203. Phone: (716) 856-9600, ext. 322. Fax: (716) 852-6086. E-mail: cody{at}hwi.buffalo.edu.

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