United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702-5011, USA
1 To whom correspondence should be addressed. E-mail: charles.b.millard{at}us.army.mil
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Abstract |
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Keywords: protein aggregation/protein engineering/ribosome inactivating proteins
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Introduction |
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Ricin, a widely available toxin from the common castor bean plant (Ricinus communis), is composed of two different protein subunits linked by a single disulfide bond. The ricin B-chain (RTB) is a lectin that binds galactose or N-acetylgalactosamine receptors on the surface of target cells to promote endocytosis and trafficking of the toxin to the trans-Golgi. The ricin A-chain (RTA) is a highly efficient N-glycosidase (kcat/Km 107 M1 s1) (Endo and Tsurugi, 1988
) that enters the cytosol of target cells by adopting a transient, metastable state to exit the Golgi (Argent et al., 2000
). Once inside the cell, RTA acts as a ribosome inactivating protein (RIP) to remove a specific adenine base of the essential 28S ribosomal RNA, effectively stopping new protein synthesis, inducing apoptosis and killing eukaryotic cells (reviewed in Lord et al., 2003
).
The potency of RTA has found beneficial application in the field of chemotherapy as a component of immunotoxin conjugates, but the toxin also has been exploited as a poison for biological warfare and bioterrorism (Franz et al., 1997). Inhalation of small amounts of ricin aerosol can rapidly and irreversibly damage cells of the respiratory tract, leading to severe pulmonary incapacitation or death (Olsnes et al., 1982
; Griffiths et al., 1996
). The lack of specific medical treatment options for ricin exposure has led to a worldwide search for effective vaccine immunogens (Hewetson et al., 1993
; Griffiths et al., 1996
, 1999
; Smallshaw et al., 2003
). Improved understanding of the immune response to RTA also may lead to new means of eliminating the systemic toxicity that has been observed during the clinical application of RTA immunotoxins (O'Toole et al., 1998
; Schindler et al., 2001
).
Past attempts to develop a ricin vaccine suggest that, although isolated RTA can induce protective immunity against the toxin in animals, the use of RTA as a vaccine component is limited by its potentially toxic RIP activity and the undesirable tendency of RTA to self-aggregate in the absence of RTB (Piatak et al., 1988). Active-site substitutions have been proposed as a means of inactivating RTA and rendering it safe for use in a ricin vaccine (Ready et al., 1991
; Roberts et al., 1992
; Smallshaw et al., 2002
). Although effective at reducing RIP activity, this approach does not address the problem of precipitation during production or storage of RTA as a biopharmaceutical. To overcome both problems simultaneously, wide-scale modifications to the RIP protein fold itself were undertaken. We report here on the conversion of RTA to a single-domain structure that is more stable than the parent molecule and demonstrate that the new scaffold suffices to produce safe and effective vaccine candidates.
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Materials and methods |
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A structurestructure alignment between RTA and pokeweed antiviral protein (PAP) was performed to determine the relative hydrophobicity of the C-terminal regions. This allowed us to assess possible differences in solubility of the individual protein chains. The electrical potentials for RTA and PAP projected on to their molecular surfaces were calculated to highlight differences in solvent polarization of the C-terminal regions. Electrostatic solvation free energies were calculated from the PoissonBoltzmann equation using a charginguncharging process (Nicholls et al., 1991; Olson and Cuff, 1999
) and the secondary structure and topology of RTA were analyzed for variations in protein compactness. Reaction field energies were determined for each protein by using a protein dielectric constant of 2, while the implicit solvent was treated by using a dielectric constant of 80.
DNA cloning
Cloning was based on the published gene sequence of RTA (Lamb et al., 1985). To prepare the RTA truncation constructs, a stop codon was incorporated at the desired position by mutagenesis. Ten amino acid residues at positions RTA 3443 were removed using the polymerase chain reaction method to produce RTA133/44198. DNA sequencing of all RTA derivatives was performed to confirm that only the desired changes had been introduced in the gene sequence. The DNA products were purified, ligated to a commercial expression vector based upon the T7 promoter system (Studier et al., 1990
) and then used to transform competent Escherichia coli BL21 DE3 cells (Invitrogen, Carlsbad, CA).
Protein expression and purification
Transformed cells were grown in TB media containing kanamycin (50 µg/ml) until reaching a cell density of 0.40.6 OD600. Induction of genes expressing RTA sequences was performed using IPTG (1 mM) at 25°C, for 1820 h.
To evaluate protein levels and the extent of aggregation during expression, cell extracts were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and immunoblotting with a previously characterized polyclonal antisera. Briefly, cells were solubilized in 50 mM sodium phosphate buffer, pH 7.3 and the resulting buffer-insoluble pellet was boiled in 6 M urea, pH 7.
Protein purification
Cell paste was dissolved in ice-cold 50 mM sodium phosphate buffer, 2 mM EDTA, pH 7.3 at a ratio of 1 g wet weight/15 ml buffer. Cells were disrupted by sonication. Homogenized cells were centrifuged using a Sorvall SS-34 rotor at 15000 r.p.m. for 15 min at 4°C. Proteins were purified from the supernatant by a combination of conventional ion-exchange separations using commercial Mono-Q and Mono-S 10/10 columns (Pharmacia, Piscataway, NJ) and hydrophobic interaction chromatography.
Characterization of purified product
Fractions containing the RTA derivatives at >95% purity as judged by Coomassie Brilliant Blue-stained gels were pooled. Buffer was changed to 120 mM NaCl, 2.7 mM KCl, 10 mM NaPO4, pH 7.4 [phosphate-buffered saline (PBS)] by dialysis and purified protein was filtered (0.2 µm pore size) and stored sterile at 4°C prior to animal studies. The identity of purified proteins was confirmed by immunoblots with polyclonal antiserum and by Edman N-terminal degradative sequencing.
As a control for comparison with the vaccine candidates, recombinant (r) RTA was expressed and purified essentially as described (O'Hare et al., 1987; Piatak et al., 1988
; Ready et al., 1991
).
Residual RIP activity
Toxic N-glycosidase activity was assessed by the ability of purified proteins to block luciferase synthesis in a commercial cell-free translation assay as described (Hale, 2001). Assays were calibrated using known amounts of purified, natural ricin (Vector, Burlingame, CA) or rRTA.
Dynamic light scattering
A Dyna-Pro MS800 instrument (Protein Solutions) was used to make right-angle dynamic laser-light scattering measurements on protein solutions at 0.8 mg/ml in PBS at 25°C. Molecular weights were estimated using the standard curve for small globular proteins contained in the Dynamics software package.
Circular dichroism (CD)
A Jasco-810 spectropolarimeter was used to take CD spectra in the far-UV region on protein solutions at 0.2 mg/ml in a stoppered 1 mm pathlength cuvette. Heating was carried out at 1 K min1 and data were not smoothed.
Protein precipitation time course
Samples of rRTA and/or RTA133/44198 at 0.2 mg/ml in PBS were incubated at 37°C and centrifuged at each time point to remove insoluble denatured protein. The total remaining soluble protein at each time point was measured by Bio-Rad protein dye assay and SDSPAGE.
Vaccine studies
A mouse model was used to evaluate the efficacy of the recombinant, RTA-derived polypeptides. Mice were vaccinated intramuscularly at 0, 4 and 8 weeks with 10 µg of the test polypeptide ± adjuvant. Three weeks after the last vaccination, blood was obtained for serology and 1 week later the mice were challenged with 10 LD50 or 5 LD50 of ricin by intraperitoneal injection or by whole-body aerosol in a class III hood line. In separate experiments, doseresponses to the vaccine candidates were determined by immunizing with increasing amounts of protein immunogen, followed by ricin challenges by whole-body aerosol or intraperitoneal injection.
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Results and discussion |
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An examination of the RTARTB interfacial region with the equivalent region of monomeric PAP revealed that the RTA sequence contains significantly more hydrophobic residues (30% versus 15%). This increase in hydrophobic character leads to an electrical potential of the RTA region that is less favorable in terms of hydration than is the corresponding PAP region (see Figure 1A). In other words, the release of water molecules at the RTA interface strongly favors the reassociation with RTB or RTA self-aggregation, whereas PAP is more hydrophilic. The notion of hydration preference was corroborated by numerical solution to the PoissonBolztmann equation for the calculation of the free energy of electrostatic charging of RTA or PAP in a low-dielectric medium surrounded by a high-dielectric solvent environment.
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Based on the globular organization and compactness of RTA, several regions in the structure were carefully evaluated for separating the two domains. The coiled linker region between residues RTA 190 and 198 was selected because: (1) it appeared hydrophilic, favoring both conformational flexibility and solvent polarization; (2) it avoided inclusion of the backbone-torsional restrictive residue, Pro200, which was expected to interfere with compact folding of the residual C-terminus; and (3) computer models of the RTARNA assembly predicted that removing the entire fragment 199267 from the RTA fold would eliminate critical residues required for high-affinity binding of the RNA substrate, including Asn209, Trp211 and Arg213 (Olson, 1997; Olson and Cuff, 1999
).
From modeling solvent effects for the truncated RTA1198, we noticed that water was now predicted to fill several protein cavities not initially exposed to solvent in RTA. One structural element of concern was a loop region (RTA 3443) which unfavorably increased overall solvent accessibility of the protein. We conjectured that removing this loop from the RTA1198 platform to create the polypeptide RTA133/44198 should further optimize compactness of the structure, thereby disfavoring protein unfolding and aggregation.
To test our hypothesis, we expressed and purified RTA1222, RTA1198, RTA133/44198 and related RTA derivatives (Figure 1C). RTA1222 was found to be less soluble during purification than were the RTA1198 truncations. Because it demonstrated the best combination of desirable features for a biopharmaceutical, we focus our discussion on the characterization of RTA133/44198 as a lead ricin vaccine construct.
Characteristics of lead candidate RTA133/44198
As predicted, neither RTA1198 nor RTA133/44198 retained toxic RIP activity as judged by failure to inhibit ribosomes in a cell-free translation assay. RTA133/44198 showed no detectable RIP activity at 10 µg/ml, under conditions where concentrations of natural ricin or rRTA at <2 ng/ml (positive controls) completely blocked cell-free translation. Loss of activity in the RTA1198 truncations is consistent with our previous thermodynamic analysis that predicted the importance of several C-terminal residues of RTA in forming the binding complex with the RNA substrate.
The purified RTA133/44198 behaved as a tightly folded, globular monomer in solution. Dynamic light scattering measurements showed that RTA133/44198 is monodisperse at 0.8 mg/ml in PBS solution at 25°C. A hydrodynamic radius of 2.11 nm was found for RTA133/44198; this radius corresponded to an apparent molecular weight of 19.3 x 103, close to the true value of 21.3 x 103.
Evidence for proper folding of RTA133/44198 was obtained by CD spectroscopy. Comparative spectroscopic analysis of the secondary structure content of RTA133/44198 corroborated that the overall structure is largely conserved from RTA. The shape of the RTA133/44198 far-ultraviolet spectrum (Figure 2A) indicated that it contains roughly the same proportion of helical and sheet secondary structures reported for RTA (Day et al., 2002); this is consistent with conservation of domain structure for the N-terminus. Proper folding of this part of the protein probably contributes to the correct presentation of the neutralizing epitope, RTA95110.
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To evaluate the practical consequences of increased thermal stability of RTA133/44198 during future vaccine production or storage, we conducted solubility experiments. Purified rRTA or RTA133/44198 was placed at 37°C and the total amount of each protein remaining in solution as a function of incubation time was measured (Figure 3A). Approximately 50% of the soluble rRTA precipitated within 48 h, with less than 10% of starting protein left in solution at 106 h. In contrast, about 60% of the RTA133/44198 remained in solution at 106 h. To visualize the extent of aggregation and to rule out contamination of either sample with proteases, we also incubated the two proteins together and analyzed soluble protein by SDSPAGE (Figure 3B).
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Comparison with other ricin vaccine candidates
Vitetta and colleagues proposed a recombinant RTA vaccine that combines active-site substitutions to reduce RIP activity with the removal of a buried vascular leak peptide (VLP) sequence (Smallshaw et al., 2002). The VLP has been implicated in a complex pulmonary vascular leak syndrome reported in cancer patients administered active RTA immunotoxins (Soler-Rodriguez et al., 1992
, 1993
; Schnell et al., 2003
). Although RTA133/44198 retains the VLP sequence, it did not cause weight loss in vaccinated animals or other signs of vascular leak syndrome. This may be explained by the elimination of RIP activity from RTA133/44198, and/or by the relatively small amounts and rapid clearance of RTA immunogens compared with RTAantibody conjugates (Smallshaw et al., 2002
). Biological activity of the buried VLP also may require RTA to partly unfold or undergo other structural reorganization dependent upon the C-terminal domain that is lacking in the RTA1198 proteins. We are currently testing this hypothesis by application of the purified RTA1198 proteins to a human endothelial cell model in vitro.
The conventional protein engineering approach of eliminating activity by introducing single amino acid substitutions can be problematic for ricin because of the resilient plasticity of the RTA active site in obtaining the catalytic transition state. For example, both Glu177 and Arg180 are invariant residues among known RIPs and are thought to play crucial roles in the catalytic mechanism (Monzingo and Robertus, 1992; Chaddock and Roberts, 1993
). Disrupting the ion pair between Glu177 and Arg180 at the RTA active site, however, often reduces but does not eliminate RIP activity. The Glu177 to Ala177 mutation demonstrated a remarkable rescue of electrostatic balance in the active site, achieved by the rotation of a proximal Glu208 into the space vacated by Glu177 (Kim et al., 1992
). Likewise, Arg180 to His180 reduces RIP activity over 500-fold, yet it remains cytotoxic (Day et al., 2002
). Additionally, as noted by Robertus and co-workers, RTA Glu177 to Gln177 retains residual RIP activity and it appears to be far less well behaved than RTA in terms of expression levels (Schlossman et al., 1989
; Ready et al., 1991
).
The RTA1198 truncations inherently lack several active-site residues, including Glu208, Asn209, Trp211 and Arg213. In separate experiments, we also attempted to inactivate RTA by single substitutions of active-site residues, including removal of the charge at positions Glu177, Arg180, Glu208 or Arg213. Consistent with previously published reports, however, we found that these proteins either were expressed at low yield or were expressed predominantly in the insoluble fraction following cell disruption (not shown). Given the poor results with expressing these individual RTA active-site mutants and the complete loss of N-glycosidase activity in RTA1198 truncations, we did not attempt to include Glu177 or Arg180 mutations in the RTA1198 platform.
Mutational and modeling studies of RTA and its binding to RNA suggest why isolated active-site substitutions often fail to produce structurally robust immunogens (see, for example, Olson and Cuff, 1999; Marsden et al., 2004
). We propose that self-organization of the native RTA tertiary fold is optimized by the electrostatic charge balance of the active-site cavity (Olson, 2001
). In other words, altering the charge balance consequently leads to structural reorganization, coupled with a reduction in stability of the protein fold. Therefore, rather than simply introducing site-specific change(s) to eliminate activity, we altered the RIP fold by dedifferentiation of the molecule to achieve a more stable structure. Because it completely lacks catalytic activity and still protects as a vaccine, the designed fold may be of wider utility in devising vaccines for other deadly RIP toxins such as abrin or modeccin.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() |
---|
Argent,R.H., Parrott,A.M., Day,P.J., Roberts,L.M., Stockley,P.G., Lord,J.M. and Radford,S.E. (2000) J. Biol. Chem., 275, 92639269.
Beaumelle,B., Taupiac,M.P., Lord,J.M. and Roberts,L.M. (1997) J. Biol. Chem., 272, 2209722102.
Carr,C.M., Chaudhry,C. and Kim,P.S. (1997) Proc. Natl Acad. Sci. USA, 94, 1430614313.
Carrell,R.W. and Lomas,D.A. (1997) Lancet, 350, 134138.[CrossRef][ISI][Medline]
Carrotta,R., Bauer,R., Waninge,R. and Rischel,C. (2001) Protein Sci., 10, 13121318.
Chaddock,J.A. and Roberts,L.M. (1993) Protein Eng., 6, 425431.[ISI][Medline]
Chaddock,J.A., Roberts,L.M., Jungnickel,B. and Lord,J.M. (1995) Biochem. Biophys. Res. Commun., 217, 6873.[CrossRef][ISI][Medline]
Chaddock,J.A., Monzingo,A.F., Robertus,J.D., Lord,J.M. and Roberts,L.M. (1996) Eur. J. Biochem., 235, 159166.[Abstract]
Day,P.J., Ernst,S.R., Frankel,A.E., Monzingo,A.F., Pascal,J.M., Molina-Svinth,M.C. and Robertus,J.D. (1996) Biochemistry, 35, 1109811103.[CrossRef][ISI][Medline]
Day,P.J., Pinheiro,T.J., Roberts,L.M. and Lord,J.M. (2002) Biochemistry, 41, 28362843.[CrossRef][ISI][Medline]
De Bernardez Clark,E., Hevehan,D., Szela,S. and Maachupalli-Reddy,J. (1998) Biotechnol. Prog., 14, 4754.[CrossRef][ISI][Medline]
Endo,Y. and Tsurugi,K. (1988) J. Biol. Chem., 263, 87358739.
Franz,D.R. and Jaax,N.K. (1997) Ricin toxin. In Sidell,F.R., Takafuji,E.T. and Franz,D.R. (eds), Medical Aspects of Chemical and Biological Warfare. Office of the Surgeon General, Department of the Army, United States of America, Washington, DC, pp. 631642.
Gorovits,B.M., McGee,W.A. and Horowitz,P.M. (1998) Biochim. Biophys. Acta, 1382, 120128.[ISI][Medline]
Griffiths,G.D., Rice,P., Allenby,A.C. and Bailey,S.C. (1996) J. Defense Sci., 1, 227235.
Griffiths,G.D., Phillips,G.J. and Bailey,S.C. (1999) Vaccine, 17, 25622568.[CrossRef][ISI][Medline]
Hale,M.L. (2001) Pharmacol. Toxicol., 88, 255260.[CrossRef][ISI][Medline]
Hammarstrom,P., Persson,M., Freskgard,P.O., Martensson,L.G., Andersson,D., Jonsson,B.H. and Carlsson,U. (1999) J. Biol. Chem., 274, 3289732903.
Hewetson,J.F., Rivera,V.R., Creasia,D.A., Lemley,P.V., Rippy,M.K. and Poli,M.A. (1993) Vaccine, 11, 743746.[CrossRef][ISI][Medline]
Jaswal,S.S., Sohl,J.L., Davis,J.H. and Agard,D.A. (2002) Nature, 415, 343346.[CrossRef][ISI][Medline]
Katzin,B.J., Collins,E.J. and Robertus,J.D. (1991) Proteins, 10, 251259.[ISI][Medline]
Kim,Y., Mlsna,D., Monzingo,A.F., Ready,M.P., Frankel,A. and Robertus,J.D. (1992) Biochemistry, 31, 32943296.[ISI][Medline]
Lamb,F.I., Roberts,L.M. and Lord,J.M. (1985) Eur. J. Biochem., 148, 265270.[Abstract]
Lebeda,F.J. and Olson,M.A. (1999) Int. J. Biol. Macromol., 24, 1926.[CrossRef][ISI][Medline]
Lord,M.J., Jolliffe,N.A., Marsden,C.J., Pateman,C.S., Smith,D.C., Spooner,R.A., Watson,P.D. and Roberts,L.M. (2003) Toxicol. Rev., 22, 5364.[Medline]
Marsden,C.J., Fulop,V., Day,P.J. and Lord,J.M. (2004) Eur. J. Biochem., 271, 153162.
McHugh,C.A., Tammariello,R.F., Millard,C.B. and Carra,J.H. (2004) Improved stability of a protein vaccine through elimination of a partially unfolded state. Protein Sci., in press.
Millard,C.B., Shnyrov,V.L., Newstead,S., Shin,I., Roth,E., Silman,I. and Weiner,L. (2003) Protein Sci., 12, 23372347.
Montfort,W., Villafranca,J.E., Monzingo,A.F., Ernst,S.R., Katzin,B., Rutenber,E., Xuong,N.H., Hamlin,R. and Robertus,J.D. (1987) J. Biol. Chem., 262, 53985403.
Monzingo,A.F., Collins,E.J., Ernst,S.R., Irvin,J.D. and Robertus,J.D. (1993) J. Mol. Biol, 233, 705715.[CrossRef][ISI][Medline]
Monzingo,A.F. and Robertus,J.D. (1992) J. Mol. Biol., 227, 11361145.[ISI][Medline]
Nicholls,A., Sharp,K.A. and Honig,B. (1991) Proteins, 11, 281296.[ISI][Medline]
O'Hare,M., Roberts,L.M., Thorpe,P.E., Watson,G.J., Prior,B. and Lord,J.M. (1987) FEBS Lett., 216, 7378.[CrossRef][ISI][Medline]
Olsnes,S. and Pihl,A. (1982) Toxic lectins and related proteins. In Cohen,P. and Van Heyningen,S. (eds), Molecular Action of Toxins and Viruses. Elsevier Biomedical Press, New York, pp. 51105.
Olson,M.A. (1997) Proteins, 27, 8095.[CrossRef][ISI][Medline]
Olson,M.A. (2001) Biophys. Chem., 91, 219229.[CrossRef][ISI][Medline]
Olson,M.A. and Cuff,L. (1999) Biophys. J., 76, 2839.
O'Toole,J.E., Esseltine,D., Lynch,T.J., Lambert,J.M. and Grossbard,M.L. (1998) Curr. Top. Microbiol. Immunol., 234, 3556.[ISI][Medline]
Piatak,M., Lane,J.A., Laird,W., Bjorn,M.J., Wang,A. and Williams,M. (1988) J. Biol. Chem., 263, 48374843.
Ready,M.P., Kim,Y. and Robertus,J.D. (1991) Proteins, 10, 270278.[ISI][Medline]
Roberts,L.M., Tregear,J.W. and Lord,J.M. (1992) Targeted Diagn. Ther., 7, 8197.[Medline]
Robertus,J. (1991) Semin. Cell Biol., 2, 2330.[Medline]
Rutenber,E., Katzin,B.J., Ernst,S., Collins,E.J., Mlsna,D., Ready,M.P. and Robertus,J.D. (1991) Proteins, 10, 240250.[ISI][Medline]
Schindler,J., Sausville,E., Messmann,R., Uhr,J.W. and Vitetta,E.S. (2001) Clin. Cancer Res., 7, 255258.
Schlossman,D., Withers,D., Welsh,P., Alexander,A., Robertus,J. and Frankel,A. (1989) Mol. Cell. Biol., 9, 50125021.[ISI][Medline]
Schnell,R., Borchmann,P., Staak,J.O., Schindler,J., Ghetie,V., Vitetta,E.S. and Engert,A. (2003) Ann. Oncol, 14, 729736.
Shin,I., Kreimer,D., Silman,I. and Weiner,L. (1997) Proc. Natl Acad. Sci. USA, 94, 28482852.
Smallshaw,J.E., Firan,A., Fulmer,J.R., Ruback,S.L., Ghetie,V. and Vitetta,E.S. (2002) Vaccine, 20, 34223427.[CrossRef][ISI][Medline]
Smallshaw,J.E., Ghetie,V., Rizo,J., Fulmer,J.R., Trahan,L.L., Ghetie,M.A. and Vitetta,E.S. (2003) Nat. Biotechnol., 21, 387391.[CrossRef][ISI][Medline]
Sohl,J.L., Jaswal,S.S. and Agard,D.A. (1998) Nature, 395, 817819.[CrossRef][ISI][Medline]
Soler-Rodriguez,A.M., Uhr,J.W., Richardson,J. and Vitetta,E.S. (1992) Int. J. Immunopharmacol., 14, 281291.[CrossRef][ISI][Medline]
Soler-Rodriguez,A.M., Ghetie,M.A., Oppenheimer-Marks,N., Uhr,J.W. and Vitetta,E.S. (1993) Exp. Cell. Res., 206, 227234.[CrossRef][ISI][Medline]
Studier,F.W., Rosenberg,A.H., Dunn,J.J. and Dubendorff,J.W. (1990) Methods Enzymol., 185, 6089.[Medline]
Villafranca,J.E. and Robertus,J.D. (1981) J. Biol. Chem., 256, 554556.
Wanker,E.E. (2000) Biol. Chem., 381, 937942.[ISI][Medline]
Weston,S.A., Tucker,A.D., Thatcher,D.R., Derbyshire,D.J. and Pauptit,R.A. (1994) J. Mol. Biol., 244, 410422.[CrossRef][ISI][Medline]
Wilkinson,D.L. and Harrison,R.G. (1991) Biotechnology (NY), 9, 443448.[ISI][Medline]
Received May 3, 2004; accepted May 10, 2004.
Edited by Amnon Horovitz