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1 Joint Graduate Group in Bioengineering, University of California, San Francisco/University of California, Berkeley, San Francisco, California 94143, USA
2 Department of Chemical Engineering, University of California, Berkeley, California 94720, USA
3 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
Reprint requests to: Douglas S. Clark, Department of Chemical Engineering, 201 Gilman Hall, University of California, Berkeley, CA 94720, USA; e-mail: clark{at}cchem.berkeley.edu;fax: 510-643-1228.
(RECEIVED December 13, 2000; FINAL REVISION June 21, 2001; ACCEPTED June 21, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/
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Abstract |
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& ß). In contrast, when proteasome assembly preceded denaturation (designated + ß) the optimum temperature was raised to a lesser degree. Moreover, the & ß and + ß preparations had apparent thermal half-lives at 114°C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild-type activity and stability thus required the proper folding of both the - and ß-subunits prior to proteasome assembly. Consistent with this behavior, dual-scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high-temperature activity. Keywords: Extremophilic recombinant enzymes; protein folding; thermostability
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Introduction |
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TOPAbstractIntroductionResultsDiscussionConcluding remarksMaterials and methodsReferences |
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The archaeal 20S proteasome represents a particularly unusual subset of known extremophilic enzymes. The proteasome structure is composed of four heptameric rings (Lowe et al. 1995) stacked to compartmentalize (Baumeister et al. 1998) 14 catalytic threonine residues (Seemuller et al. 1996). The large barrel structure is responsible for processive degradation of cytosolic proteins (Akopian et al. 1997) and is physiologically relevant to the heat-shock response of thermophilies (Reupp et al. 1998). The archaeal proteasome is believed to be the precursor of the ubiquitin/proteasome protein degradation pathway in eukaryotes (Zwickl et al. 1991,1992a).
Since the 1989 discovery of the proteasome complex from the moderate thermophile Thermoplasma acidophilum (Ta) (Dahlmann et al. 1989), wild-type proteasomes have been isolated and characterized from the moderate thermophile Methanosarcina thermophila (Mt) (Maupin-Furlow and Ferry 1995), the hyperthermophile Pyrococcus furiosus (Pf) (Bauer et al. 1997), and the extreme thermophile Methanococcus jannaschii (Mj) (Michels and Clark 1997). The 20S proteasome from Ta was the first proteasome overexpressed in E. coli (Zwickl et al. 1992b), and several papers have since appeared describing various aspects of that proteasome's structure, activity, and assembly (Dahlmann et al. 1992; Zwickl et al. 1994; Lowe et al. 1995). Recently, recombinant proteasomes from Mt (Maupin-Furlow et al. 1998) and Mj (Wilson et al. 2000) were also produced in E. coli, allowing extensive investigation of proteasome activity. Gene comparisons indicated phylogenetic relationships between each of these proteasomes and the Ta proteasome.
Previous studies of the recombinant 20S proteasome from Mj showed that the proteasome requires a nucleotidase complex such as PAN (a proteasome-activating nucleotidase) to mediate the energy-dependent hydrolysis of folded-substrate proteins (Wilson et al. 2000), which is believed to be a central activity in the elimination of misfolded and defective proteins by the proteasome within the cell. The recombinant Mj proteasome did hydrolyze ß-casein in the absence of ATP, however, presumably because the casein was significantly or completely denatured at most of the assay temperatures (which ranged from 35–100°C). Proteolytic activity of the recombinant Mj proteasome towards ß-casein increased to at least 100°C, but was not investigated beyond this temperature. Nor were comparisons made between the activity or stability of the recombinant and native proteasomes.
Previous comparisons of wild-type thermophilic proteins and their recombinant counterparts expressed in mesophilic hosts have revealed, in some cases, significant differences in activity (Yamamoto et al. 1999; Lee 2000; Porcelli et al. 2000) and stability (Cacciapuoti et al. 1999; Porcelli et al. 2000). Such discrepancies are not surprising considering the dramatic differences in the folding environments experienced during protein expression in an extreme thermophile relative to a mesophile. Herein we report that expression of Mj proteasome subunits in E. coli did not produce the native structure, as evidenced by compromised enzyme activity and stability. Although the suboptimally folded Mj proteasome was active, denaturation of the individual subunits followed by refolding and assembly at elevated temperatures was required to achieve wild-type activity and stability. This result has important implications for the expression and folding of other recombinant extremophilic enzymes in mesophilic hosts, and may help to elucidate molecular interactions and structural motifs responsible for enzyme activity and stability in extreme environments.
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Results |
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31 and 25 kD when analyzed by SDS-PAGE. No N-terminal sequence was detected for the 31-kD band, but N-terminal sequencing of the 25-kD band revealed an N-terminal sequence (TTTVGLI_DDA) corresponding to the ß-subunit of the 20S proteasome (Bult et al. 1996). Internal sequencing and peptide mapping of the 31-kD band provided a 14-residue sequence (LTYGEEISIEMLAK) matching residues 102–115 of the Mj 20S proteasome -subunit.
Recombinant protein production and purification
Genes encoding the proteasome subunits were cloned individually into two separate cell lines that allowed for investigation of holoenzyme assembly. Freshly transformed cells were required for efficient expression of each subunit. Storage of inoculation culture overnight resulted in a fivefold decrease in expression of the ß-subunit and use of frozen inoccula gave no ß-subunit expression upon induction. No proteolytic activity was observed in 1:1 stoichiometric mixtures of - and ß-subunit lysates incubated at room temperature or 37°C. Native PAGE indicated that seven-membered -rings did form at 37°C; however, there was no evidence of ß-ring assembly or fully assembled proteasome (data not shown). However, heating 1:1 mixtures of - and ß-subunit lysates at temperatures of 70–85°C for 15–120 min produced proteolytically competent proteasome. The heating process induced the assembly of proteasome complexes while simultaneously reducing the overall level of contaminating E. coli host-cell proteins (Fig. 1
). Optimal specific activity of active recombinant proteasome (based on the amount of total protein) was observed after continuous heating at 70°C for 2 h.
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). To ensure that the primary sequence of the recombinant protein corresponded to the published gene sequence, recombinant plasmids were sequenced, revealing total complementarity to the native sequence.
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+ ß configuration was prepared by combining crude lysates of - and ß-subunits followed by incubation at 70°C. The + ß sample heat treated (HT) for 45 min is referred to as HT70°C. Additional preparations were denatured in urea following the initial incubation, and dialzyed at either 75°C or 85°C. Another type of preparation, denoted & ß, consisted of a 1:1 stoichiometric combination of - and ß-subunits individually heat purified, followed by urea denaturation and dialysis. The nomenclature used to describe the sample preparations consists of the configuration followed by the urea concentration and the subscripted dialysis temperature (e.g., & ß 0M75°C). The different preparations are summarized in Table 1.
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+ ß preparations at 70°C resulted in assembled, functional proteasome prior to denaturation at room temperature with urea. In contrast, treatment of the & ß sample with 4 M urea resulted in denaturation of proteasome subunits prior to the formation of assembled, functional proteasome. Assembly of & ß proteasomes occurred during subsequent high-temperature dialysis. A screen of postdialysis proteasome activity at urea concentrations ranging from 0–8 M identified an optimum at 4 M urea for both the + ß and & ß preparations.
Denaturation with 4 M urea and high-temperature renaturation caused a shift in Topt for both preparations. Urea-denatured + ß renatured at 75°C ( + ß 4M75°C) still had maximum activity at 95°C; however, significant activity remained at temperatures up to 115°C (Fig. 3A). The urea-denatured & ß sample renatured at 75°C ( & ß 4M75°C) showed a significant shift of the Topt to 110°C with a concomitant decrease in specific activity. As the renaturation temperature was increased to 85°C, Topt of both the + ß and the & ß preparations shifted to still higher temperatures (Fig. 3B). A bimodal activity distribution was observed for several samples. The optimal temperature for the & ß 4M85°C sample was 115°C, comparable to the native enzyme.
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90% for + ß 0M85°C and & ß 0M85°C to 95% for + ß 4M85°C and & ß 4M85°C (SDS PAGE results, not shown). Nondenaturing PAGE of pre- and post-HPLC-purified recombinant samples showed several contaminating proteasome complexes (Fig. 4). -Ring duplexes [2(7)] and single -rings [7] accompanied all fully assembled recombinant proteasome samples [2(7) + 2(ß7)]. ß-Subunit trimers [ß3] were noted only in the prepurified HT70°C sample. Band identities were verified by running SDS PAGE in a second dimension to the native PAGE gels (data not shown).
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+ ß 4M85°C, and & ß 4M85°C, two additional samples were prepared to evaluate refolding contributions by each subunit to stability and Topt. These combinations consisted of a 4 M–urea-denatured/85°C-renatured subunit combined with a 70°C heat-purified subunit. The sample mixture was then incubated at 70°C to initiate assembly. These samples are designated as den/ren + ßHT and HT + ßden/ren. The Topt of den/ren + ßHT was similar to that of + ß 4M85°C (data not shown), whereas the combination HT + ßden/ren exhibited no proteolytic activity.
Native PAGE of the proteasome den/ren + ßHT provided results similar to what was observed for + ß 4M85°C with the addition of a band in the ß-trimer region. A native gel of HT + ßden/ren showed no intact proteasome and an abundance of -ring duplexes, which migrated at a slightly higher molecular weight, indicating a possible difference in molecular shape and/or charge.
Recombinant proteasome thermal inactivation
Thermal inactivation trajectories of recombinant proteasome samples are shown in Figure 5. Thermal half-life values (t1/2) were calculated assuming a first-order exponential decay of proteasome activity. Thermal inactivation was conducted at pH 7.5 and pH 5.7 at 114°C. At pH 7.5, t1/2 for & ß 4M85°C was 54.2 ± 3.6 min, which was higher than t1/2 for either + ß 4M85°C (26.2 ± 1.5 min) or HT70°C (11.6 ± 3.0 min) (Fig. 5A). As the pH was lowered to 5.7, t1/2 decreased for + ß 4M85°C to 6.47 min (an 88% reduction) and for HT70°C to 4.94 min (a 57% reduction). By comparison, the t1/2 for & ß 4M85°C decreased by only 25% to 41.2 min (Fig. 5B).
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108°C (data not shown), slightly higher than the observed Tm for both + ß 4M85°C (104°C, Fig. 6A) and & ß 4M85°C (104°C, Fig. 6B). Following the initial melting transition, samples were cooled to allow for subsequent reassembly of the proteasome. As the sample was reheated to 144°C, there was no discernible transition or indication of melting of any refolded protein components in the previously observed melting region (80–110°C).
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den/ren + ßHT and HT + ßden/ren, were also analyzed by the high-temperature melting DSC protocol. The Tm of den/ren + ßHT was 103°C. The inactive HT + ßden/ren, which consisted primarily of -rings and -ring duplexes, exhibited a Tm of 97.3°C. As with the other preparations, no indications of reassembly were observed.
Another set of DSC experiments, termed the "DSC reassembly" experiments raised the initial sample temperature to 108°C before initiating the cycle of cooling and reheating to 144°C. In this case, DSC signals were observed in the melting region (80–110°C) during the reheating portion of the temperature profile, indicating that the thermally denatured proteasome had reassembled following its initial excursion to 108°C. The Tm values for the + ß and & ß preparations were both 105°C (Fig. 6
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Discussion |
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The physiological substrates of the proteasome complex are completely unfolded (linearized) polypeptide chains. Denatured proteins are translocated to the active site core by an unknown mechanism to be cleaved into smaller (6–10 residue) pieces in a processive manner (Kisselev et al. 1998). The specificity indicated by screens of small colorimetric or fluorometric substrates may thus have little or no relevance when identifying specificities for protein substrates. Such results may thus be misleading when considering cleavage specificities for protein substrates. However, synthetic colorimetric substrates do provide an excellent marker of proteasome activity. In our case, a colorimetric (p-nitroanilide) substrate was used because it has exceptionally high thermal stability (Michels and Clark 1997) and is compatible with visible wavelength spectrophotometric activity assays.
N-terminal sequencing of purified proteasome subunits revealed that the -subunit was unsequencable. The cause for this observation has not been identified. Peptide mapping and internal sequencing was thus required to verify the identity of the native -subunit. Zwickl et al. (1992b) observed that the native -subunit of the Ta proteasome was similarly unsequenceable, whereas the recombinant protein provided an unambiguous N-terminal sequence. This difference may arise from posttranslational modification of the native enzyme in vivo.
Unlike other recombinant archaeal protein complexes studied to date, assembly of the 28-mer complex from Mj was highly temperature dependent. Native PAGE and proteolytic activity experiments indicated the absence of assembled proteasomes for samples incubated at 37°C. The lowest temperature compatible with active proteasome production was found to be 50°C, with an optimal assembly temperature of 70°C. The requirement for high-temperature enzyme assembly provided a significant advantage for purification of the proteasome complex, as most E. coli proteins precipitated out after heat treatment. The total protein specific activity increased as a result of increased proteasome assembly coupled with the reduction of host cell impurities. The purity level of heat-treated samples was about 80–90% based on quantitation of Coomassie stained SDS PAGE gels. The major contaminant of recombinant Mj proteasome preparations was unincorporated -ring duplexes. The presence of duplexes was verified by native PAGE 2D electrophoresis (data not shown). Addition of higher concentrations of ß-subunits was not sufficient to titrate -ring duplexes to active proteasomes (data not shown). Scanning electron micrographs (SEM) of recombinant Ta proteasomes also revealed the presence of a high concentration of -ring duplexes and a minor population of -ring monomers (Zwickl et al. 1994).
Enzyme assembly achieved by optimal heat treatment yielded proteasome with a Topt of 95°C, similar to that of proteasome from the moderate thermophile Ta (Dahlmann et al. 1992). Activity–temperature profiles comparable to the native Mj enzyme (Fig. 2) were achievable only by urea denaturation and high-temperature refolding. As dialysis temperatures approached the optimal growth temperature for Mj (ca. 86°C), optimal subunit folding and/or holoenzyme assembly was obtained. However, the Topt value of + ß 4M85°C and + ß 0M85°C did not vary as dramatically as Topt of the & ß preparations (Fig. 3). Preassembled proteasome ( + ß) thus appeared to be resistant to the denaturation and refolding events that enabled the observed shift to the highest temperature optimum. Moreover, the Topt of & ß 4M85°C shifted to a higher temperature than & ß 0M85°C, indicating that elevated temperature alone was not sufficient to achieve a Topt similar to the native enzyme. A schematic representation of the proteasome assembly under different conditions is presented in Figure 7.
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-subunits were combined with heat-treated ß-subunits and vice versa. No activity was obtained for the HT + ßden/ren sample, whereas the behavior of the den/ren + ßHT sample was comparable to + ß 4M85°C. The ß-subunit of Ta has been observed to aggregate when isolated in situ (Zwickl et al. 1994). Likewise, the Mj ß-subunit may not remain soluble during the renaturation step in the absence of the -subunit. Interestingly, ß-subunits appeared to be somewhat stable to denaturation/renaturation in the presence of -subunits, as evidenced by the production of active proteasomes during the preparation of & ß 4M85°C. Assembled - rings may thus serve as a site-specific template for proper ß-subunit folding. Monomeric ß-subunits may also be stabilized as they stack on -ring scaffolds by simple mass action. The apparent stability and spontaneous low-temperature assembly of -rings implicates ß-subunit folding and/or stacking as the limiting determinant of native activity. However, when the den/ren + ßHT combination sample was assayed for thermostability and Topt, the results mirrored the subnative performance of + ß 4M85°C.
Comparison of the temperature–activity profiles of & ß 0M85°C and & ß 4M85°C indicated that only high-temperature refolding of individual - and ß-subunits allowed for a Topt equal to that of the native enzyme. Subunit folding under suboptimal low-temperature conditions experienced during expression in a mesophilic host, followed by high-temperature proteasome assembly, may "lock" the ß-subunit into a relatively labile conformation. Improperly folded ß-subunits could, therefore, contribute to the drastic reduction in t1/2 and lower Topt of + ß 4M85°C and the subnative Topt for den/ren + ßHT. Concerted ß-subunit folding on an -ring scaffold may be required to achieve the optimal folded conformations required to maximize the number of stabilizing protein–protein interactions. A similar phenomenon was observed during the assembly of the trp repressor dimer (Schevitz et al. 1985), in which case -helix domains from each monomer must be entwined to form the stable complex.
DSC analysis was used to obtain additional insights into the underlying differences in the various proteasome preparations. At first glance, the DSC data appear to conflict with the themostability data. The observed Tm values (105°C) obtained from the high-temperature melting experiments are about 10°C lower than the temperature (114°C) used for the thermal half-life determinations. In particular, a t1/2 of 54 min for & ß 4M85°C would seem to be irreconcilable with a Tm for the same sample of 105°C. This conundrum can be explained with the results from the refolding DSC experiments. As the DSC sample temperature increased to 108°C, the temperature was high enough to induce reversible disassembly of the proteasome's quaternary structure without irreversibly denaturing individual subunits. During the temperature ramp-down to 20°C, the proteasome reassembled, thus enabling the subsequent measurement of Tm. The data therefore suggest that the proteasome is able to reassemble when temperatures reach 108°C but not once the temperature exceeds 144°C. The thermal inactivation experiments were conducted at 114°C and the samples immediately cooled before residual activity was measured. The proteasome was able to reassemble even after exposure to 114°C, and the resulting thermal inactivation profiles did not reflect the t1/2for an irreversible denaturation but instead measured the efficiency of reassembly following a high-temperature exposure.
Reevaluation of the inactivation data in this light reveals that & ß 4M85°C regained an active conformation much more readily than the other preparations. Exposure to 114°C did not irreversibly denature subunits prepared in the & ß scenario; however, proteasome subunits prepared under different conditions were destabilized to a higher degree, and were therefore less competent to reassemble. This again underscores the inherent stability of the & ß 4M85°C subunits and their proficiency for reassembly. This ob-servation may have physiological relevance with respect to the role of the proteasome as an essential heat shock protein. The inherent ability of the molecule to reassemble following a transient temperature increase may provide Mj with an evolutionary advantage over its mesophilic counterparts.
It is evident that attainment of wild-type activity and stability for the Mj proteasome requires the proper folding of both the - and ß-subunits prior to proteasome assembly. This observation may be representative of potential problems with the expression of thermophilic enzymes in mesophilic hosts. For example, in the case of cyclodextrin glucanotransferase (CGTase) from the thermophile Thermococcus Sp B1001, CD and DSC differences were observed between heated and unheated recombinant enzyme preparations. Despite the heat treatment, the characteristics of the recombinant CGTase remained different from those of the native enzyme (Yamamoto et al. 1999). Likewise, the Topt of -glucosidase cloned from thermophilic Bacillus sp. DG0303 was lower than that of the native enzyme (Lee 2000), as were the thermoactivity and thermostability of 5'-methylthioadenosine phosphorylase (MTAP) cloned from the extreme thermophile Sulfolobus solfataricus (Ss) (Cacciapuoti et al. 1999). In the case of MTAP, improper disulfide bond formation was responsible for the altered properties of the recombinant enzyme.
The MTAP example above exemplifies disulfide scrambling, one common folding discrepancy encountered in recombinant proteins expressed in E. coli. However, in the case of the Mj proteasome observed shifts in thermostability and Topt were not due to the accurate reformation of disulfide bridges. The proteasome has no observed disulfide bonds; thus, the increased stability and thermoactivity of the denatured/renatured & ß proteasome is due to the optimization of noncovalent inter- and intramolecular interactions.
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Concluding remarks |
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Denaturation of recombinant proteosome subunits and high-temperature renaturation and assembly were absolutely required to match the thermostability and Topt of the native enzyme. Although an example of partial proteasome denaturation resulting in enhanced activity of eukaryotic rat muscle proteasomes has been reported previously (Dahlmann et al. 1993), the novelty of the recombinant Mj proteasome system lies in the ability to produce two active conformers with significantly different activity profiles and thermostabilities. Detailed characterization of these conformers will enable the elucidation of structural motifs and interactions required for maximum temperature activity and stability of the proteasome. Crystal structure information could clarify, for example, the relative importance of ionic versus hydrophobic interactions responsible for enzyme function and stability in extreme environments. Identification of modified inter- and intrasubunit–subunit interactions will provide targets for stability enhancement experiments, and may lead the way for engineered thermostable enzymes.
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Materials and methods |
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Proteolytic activity assay
Proteolytic activity was measured by hydrolysis of CBz-Ala-Ala-Leu-pNA (Boeringer-Mannheim) on an Aviv Model 14NT-UV-VIS spectrophotometer fitted with a magnetically stirred, pressurized Peltier-heated cell holder; 1.37 mL of 1X Zymogram Developing Buffer (Invitrogen) was heated for 45 sec to 2 min in a 3-mL quartz cuvette with a Teflon plug. Eighty microliters of 5 mM CBz-AAL-pNA in dimethyl sulfoxide (DMSO) were added, and the sample chamber was sealed and pressurized to 40 psi with N2. After a total incubation time of 5 min, which separate direct temperature measurements had shown was sufficient for the assay solution to reach the desired temperature, 50 µL of enzyme sample was added via a 500-µL Hamilton syringe fitted with a no-slip dispenser. The absorbance was measured at 405 nm (A405 nm) over a 60-sec period to obtain activity trajectories, from which initial rates were calculated. The measured extinction coefficient for pNA was 10,500 M-1 cm-1. Activity–temperature profiles of activity values measured at different temperatures were constructed using buffers adjusted to maintain the pH at 7.5 at all assay temperatures.
Electrophoresis, peptide sequencing, and protein identification
Native PAGE experiments utilized 4–12% tris-glycine gels and high molecular weight (HMWT) calibration standards (Amersham-Pharmacia Biotech). Overall purity of the proteasome was determined by densitometry measurements conducted on scanned images of Coomassie Blue-stained gels. Samples purified for sequencing were transferred to a sequencing grade polyvinyldifluoride (PVDF) membrane from an SDS-PAGE gel (Biorad) at 50 V, 135 mA, for 1 h in a Novex XLII Blot Module. The membrane was stained with amido black, and two prominent bands at 26 and 31 kD were excised and submitted to the UC Davis Protein Structure Laboratory for internal and N-terminal sequencing. Sequencing results were entered into the Methanococcus jannaschii Genome Database at http://www.tigr.org.
Gene cloning and recombinant protein production
Restriction endonucleases and other cloning enzymes were purchased from New England Biolabs. The genes encoding the proteasome - and ß-subunits were cloned via sticky end PCR from genomic Mj DNA into separate pET-21a (Novagen) expression vectors. The overhang regions of the primers form 5' NdeI and 3' BamHI sticky ends. Cloning was performed as described previously (Kim et al. 1997). BL21 (DE3) E. coli cells (Novagen) containing the pSJS1240 vector (Zeng 1998) for expression of rare Archaeal tRNAs were transformed with pET-21a vectors containing the and ß genes. Selection of viable colonies using 100 mg/mL ampicillin and 60 mg/mL spectinomycin on Luria-Bertani (LB) agar plates was completed at 37°C overnight. Plasmid presence for each cell line was accomplished with 0.7% agarose/TBE gels run at 85 V for 65 min. Fresh transformants were produced prior to each fermentation to ensure high expression levels. Fifteen liters of enhanced EzMix LB broth (Sigma) were sterile filtered into a 20-L Nalgene Biovessel fitted with a perforated silicone tube ring sparger. Upon growth of the cells to A600 nm = 1.0, sterile IPTG was added to 1 mM. After 3 h, the cells were centrifuged (5,000 rpm, 4°C), and the cell pellet was frozen in liquid nitrogen and stored at -80°C.
Purification of recombinant Mj 20S proteasome
Cell lysis and clarification were performed as with the native enzyme. The + ß configuration was prepared by first combining crude lysates of - and ß-subunits followed by incubation at 70°C for 15–90 min. Samples heat treated (HT) for 45 min are referred to as HT70°C. Precipitated E. coli proteins were removed by centrifugation at 19,000 rpm for 15 min. The supernatant was concentrated on a 30-kD Biomax membrane in an Amicon stirred cell.
Protein denaturation and refolding
Two configurations of heat-purified proteasome preparations were utilized for refolding experiments. The first configuration was identical to the + ß preparation described above. The second preparation, denoted & ß, consisted of a 1:1 combination of - and ß-subunits individually heat purified. To effect denaturation, 0–4 M urea was added to heat-purified samples of each configuration and incubated at room temperature for 1 h. Protein concentrations in these experiments ranged from 1–2 mg/mL. Samples were renatured by dialysis (10 K Snakeskin Dialysis Tubing, Pierce) for 90 min against buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, pH20°C = 8.19, pH85°C = 7.5) at 75°C or 85°C. The dialysate was centrifuged (14,000 rpm, 20°C) to remove precipitates, and except where noted, the supernatant was used for subsequent analyses of the recombinant protein. The nomenclature used to describe sample preparations will appear as sample configuration followed by the urea concentration and the subscripted dialysis temperature (e.g., & ß 0M75°C).
HPLC purification of recombinant preparations
Refolded proteasome preparations were concentrated in 30-kD Centriprep (Millipore) devices. Two and one-half milliliters of concentrated sample were loaded onto a 105-mL (30 x 2.12-cm) Biosep-SEC-S 4000 preparative HPLC column (Phenomenex) equilibrated with Buffer C (Buffer A with 300 mM NaCl). Fractions absorbing at 215 nm were collected, concentrated to 1–2 mg/mL, and assayed for protease activity at 95°C. Maximum Cbz-Ala-Ala-Leu-pNA hydrolysis corresponded to a peak with a retention time of 21–23 min, which correlated to the molecular weight of thyroglobulin (669 kD) based on HMWT calibration standards (Amersham Pharmacia Biotech). This fraction contained between 17 ( & ß 4M85°C) and 69% (HT70°C) of the precolumn proteasome activity, and was used to determine the thermal half-life (t1/2) for each sample configuration.
Thermal inactivation
Seventy-microliter samples of HPLC-purified recombinant proteasome (140 µg total protein/mL) were aliquoted to borosilicate glass microvolume HPLC vial inserts (Hewlett-Packard) and stoppered with rubber plugs. Each insert was placed into a crimp-top HPLC autosampler vial, crimped, and incubated at 114°C. Aliquots were removed at 12-min intervals and assayed by the standard procedure at 95°C.
Dual scanning calorimetry (DSC) of recombinant preparations
Recombinant proteasome samples were concentrated to 16–40 mg/mL in 30-kD Centriprep (Millipore) units and diafiltered with five volumes of DF buffer (50 mM HEPES, 200 mM NaCl, pH 7.5). 30–40 µL of concentrated sample was placed in silver DSC sample pans and hermetically sealed with a pan press. DF Buffer was used as a reference. Samples were assayed on a Seiko Instruments DSC120 differential scanning calorimeter (Japan). Each experiment consisted of an initial melting phase of increasing temperature followed by a refolding period of decreasing temperature followed by another high-temperature melting phase. Experiments conducted to identify proteasome melting temperatures (referred to as high-temperature melting experiments) were conducted with a thermal profile of 20°C to 144.2°C to 20°C to 144.2°C at a scan rate of 2°C/min. Experiments performed to investigate reassembly of thermally dissociated proteasomes (referred to as DSC reassembly experiments) utilized a temperature profile of 20°C to 108°C to 20°C to 144.2°C at the same scan rate. Data were analyzed with UNIX-based Seiko software.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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A405/sec) of recombinant Mj crude lysates heated at 60°C, 70°C, and 80°C for 0–180 min. Total protein values are represented as 60°C (filled squares), 70°C (filled triangles), and 80°C (filled circles). Activities measured by the standard protocol at 95°C are represented as open versions of the symbols above. Errors for protein assays were calculated from duplicate samples. Error bars for activity assays of 70°C samples represent one standard deviation for triplicate assays.
