1 Genomics Institute of the Novartis Research Foundation, 3115 Merryfield Row, San Diego, CA 92121 3 The Scripps Research Institute, Department of Molecular Biology, La Jolla, CA 92037, USA
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Abstract |
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Keywords: protein chaperone/protein folding/recombinant gene expression/solubility assay
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Introduction |
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A major source of unfolded proteins in vivo is the nascent chain of translating genes. Gene expression in response to translational stress is related to the heat-shock response (Parsell and Sauer, 1989), but has not been closely examined. Genome arrays used to study global gene expression in E.coli at elevated temperature (Richmond et al., 1999
) show changes in a large number of transcripts. To study translational misfolding rather than heat stress, we used recombinant expression of folded and misfolded proteins as a stimulus. Our results indicate a unique set of genes respond to translational misfolding. The nature of these genes implies that many known heat-shock and chaperone proteins, as well as ribosome-associated proteins suggestive of translational regulation, are induced to deal with translational stress.
We have applied these results in a practical way by developing a screen to improve our ability to generate recombinant proteins for structural studies. Misfolded protein often appears as insoluble aggregates when overexpressed in E.coli. Several recent approaches have been used to detect soluble recombinant protein. They utilize protein fusion to a reporter gene such as CAT (Maxwell et al., 1999), GFP (Waldo et al., 1999
), or lacZ
(Wigley et al., 2001
) as a measure of the amount of soluble protein expressed. Though convenient, these reporters have the potential to be biased by the nature of the fusion. The reporter portion of the fusion may alter the solubility of the target protein, either positively or negatively, giving unexpected results when expressing in the absence of the reporter fusion. By utilizing a sensory and regulatory system already existing in the host, we have created a general screen for detecting misfolded, and typically insoluble, recombinant proteins without the requirement of a direct protein fusion. We have also demonstrated the utility of this approach combined with mutagenesis to create soluble fragments of recombinant proteins in E.coli. The approach provides a general means of providing folded domains for structural studies.
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Materials and methods |
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Clones expressing properly folded or misfolded human proteins were obtained from the GeneStorm collection (Invitrogen). Clones containing the Unigene accession numbers L35545, U18291, M94856, M22146, D87116, M63167, M68520, M60527, M36881, M36981, U35003, S79522, X73460, D14520, U14968, M86400 were provided in the pBADThio vector to provide arabinose-inducible expression. Thermotoga maritima genes were amplified from genomic DNA and cloned into the expression vector pMH1 which encodes a 12 amino acid N-terminal tag containing a 6X-histidine repeat for purification and detection. Reporter vectors were constructed by insertion of a PCR amplifer of 300 bp upstream of the ibpAB, ybeD, yhgI or yrfGHI genes upstream of ß-galactosidase in a pACYC184 derivative.
Rep68 was cloned from ATCC 68066 containing the entire genome of the human adeno-associated virus 2 (AAV2). Putative domains comprised of bases 1646, 6471456 and 14571611 were amplified from the full-length template and cloned into pMH1. The above template was also used in amplifications of the full-length gene for fragmentation. Two micrograms of the Rep68 amplifer were used in each of five fragmentation reactions containing 1, 0.1, 0.01, 0.001 or 0 U DNase I (Boeringer Mannheim) as well as Pfu polymerase and dNTPs. Reactions were set up on ice with the DNase added immediately prior to temperature cycling in an MJ Research thermocycler according to the following: 10 min at 25°C, 15 min at 95°C and 30 min at 72°C. Each reaction was run on a 1% agarose gel and fragments corresponding to 16001000, 1000850, 850600 and 600300 bp were extracted. Each pool was used as above for blunt cloning and ligation into pMH1 as above and introduced into the reporter cell line HK 57 for screening.
Thermotoga proteins used for expression studies evaluating proteins of known expression characteristics were cloned into pMH1 as described above. Coding regions were introduced for: TM0560, TM0414, TM0574, TM0703, TM0554, TM0556 (soluble expression); TM0688, TM0633, TM0712, TM0343, TM0218, TM0294 (mixed expression); TM0289, TM0564, TM0540, TM0425, TM0731, TM0413 (insoluble expression).
Cell growth and protein expression
Escherichia coli strains MG1655 (F- lam rph1) and KY1429 (F-araD139 (argF-lac)169 lam flhD5301 fruA25 relA1 rpsL150 zhh50::Tn10 rpoH606(ts) deoC1) were transformed with expression plasmids encoding M36881 [human lymphocyte-specific protein tyrosine kinase (LCK)] or M86400 [human phospholipase A2 (PLA)] for expression profiling. Cells were cultured at 37°C in Luria broth (LB) containing ampicillin. Protein expression was induced by the addition of L-arabinose to a final concentration of 0.1% for 1 h. KY1429 cells were cultured as above except initial growth was performed at 32°C followed by a shift to 42°C for non-permissive expression of rpoH606. Top10 cells (F- mcrA
(mrr-hsdRMS-mcrBC)
80lacZ
M15
lacX74 deoR recA1 araD139
(ara-leu)7697 galU galK rpsL endA1 nupG) containing the ibpAB promoter fusion (pHK57), were transformed with expression constructs listed above. ß-Galactosidase assays were performed essentially as described by Miller (Sambrook et al., 1989
). Fractionation of soluble and insoluble proteins was performed by centrifugation. Cultures were resuspended in 50 mM Tris pH 7.9, 50 mM NaCl, 1 mM MgCl2, 3 mM methionine and sonicated for 2 min on ice. Cell debris and insoluble protein aggregates were pelleted by centrifugation at 3000 g for 15 min. The soluble fraction was removed and the pellets resuspended in an equivalent volume of lysis buffer.
Probe preparation and hybridization and analysis of labeled mRNA
Labeled mRNA was prepared and hybridized to an E.coli whole genome array (Affymetrix) essentially as described previously (Lockhart et al., 1996; Wodicka et al., 1997
). This gene chip contains 25-mer oligonucleotide probes for each of the 4290 known E.coli genes. Standard Affymetrix GeneChip analysis software was used to measure individual gene expression and to perform pairwise comparison of gene expression levels for pre-induction and post-induction samples. Comparisons of changes in gene expression for properly folded and misfolded genes were analyzed for individual gene probe sets.
Microplate solubility screening
Ninety-six-well microplates containing 200 µl of LB with 100 µg/ml ampicillin and 34 µg/ml chloramphenicol were inoculated with single colonies from above and grown overnight with shaking at 37°C. Overnight cultures were used to inoculate 200 µl of the same media and incubated at 37°C until reaching an average OD600 of 0.5. Cultures were induced with a final concentration of 0.2% arabinose. After 30 min, a cocktail of ceftriaxone and cefotaxime was added to each well to a final concentration of 10 µg/ml of each and the plates were incubated for an additional 1.5 h. These antibiotics provide a convenient method of lysis in microplate format without the need to add detergents that might solublize misfolded protein. Cultures were harvested after 2 h total of induction by centrifugation at maximum speed for 15 min to pellet cell debris on the bottom of the wells. The soluble lysate was then separated by transferring 25 µl into one set of clean microplates for ß-galactosidase activity screens and 75 µl into Nunc Maxisorp ELISA plates for Ni-HRP screening.
ß-Galactosidase activity screening of lysates was performed using a variation of the Miller protocol (Maxwell et al., 1999). A 50 µl aliquot of 4x Z-buffer and 50 µl of 4x ONPG were added to microplates containing 25 µl of soluble lysate. After development of yellow color in positive control wells, the reaction was quenched with 75 µl of 1 M Na2CO3 pH 8. The A420, A550 and reaction times were recorded and used along with the OD600 data to calculate ß-galactosidase activity (Maxwell et al., 1999
).
Ni-HRP screening was performed similar to an ELISA. A 75 µl aliquot of lysate plus 25 µl TBS was bound overnight at 4°C to a microtiter plate and blocked with 1% (w/v) BSA in TBS for 4 h at 25°C. Plates were then washed 3x with TBST and 100 µl of Ni-HRP conjugate (KPL Labs) was added at a dilution of 1:2500 and incubated 1 h at 25°C. The plates were then washed with TBST and 100 µl of the HRP substrate (KPL Labs) was added and color was allowed to develop until the positive control well was deep blue. The reaction was quenched with 100 µl of 1 N HCl and the A420 determined. Solubility scores were calculated by weighting the Ni-HRP A420 readings such that the experimental mean was one order of magnitude greater than the mean of the ß-galactosidase activity scores, then dividing the Ni-HRP absorbance by the ß-galactosidase activity. This calculation was found empirically to provide good distinction between soluble and insoluble proteins.
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Results |
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To examine gene expression as a result of misfolded protein, representative genes were cloned as fusion proteins to thioredoxin under control of the tightly regulated arabinose promoter. PLA is almost entirely soluble, as determined by cell lysis and fractionation by centrifugation (Figure 1). Further evidence of proper folding of this protein was obtained through dynamic light scattering of purified protein and the ability to crystallize it from a single affinity purification step (unpublished data). Under equivalent expression conditions, LCK is expressed almost exclusively as insoluble protein. Both proteins were expressed at sufficient levels to be the predominant translation product. mRNA preparations from induced and non-induced cultures were prepared and used to probe for gene expression.
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Not surprisingly, many of the genes induced by translational misfolding have known chaperone activity. These include the well characterized dnaJ, dnaK and grpE genes. The corresponding proteins interact as a complex with misfolded or denatured protein in an ATP-dependant repair process. Likewise, mopAB genes forming the GroELS folding repair complex are induced under translational misfolding conditions. IbpAB are small heat-shock polypeptides associated with inclusion body aggregates of recombinant protein (Allen et al., 1992). Whereas they do not appear to behave as folding chaperones directly, they bind misfolded protein and interact with the DnaJK GrpE proteins as a chaperone system (Thomas and Baneyx, 1998
). Hsp33, the gene product of the yrfI gene was recently identified as a chaperone protein responsive to oxidizing conditions (Veinger et al., 1998
). Genes implicated in degradation of denatured protein are also induced by translational misfolding. The lon, clpBP and hslUV protease genes are expressed at increased levels. Under normal cell growth these proteases serve an important recycling function. Insoluble aggregates are relatively resistant to proteolysis and this recycling pathway is ineffective for recombinant protein expression.
Induction of ribosome-associated genes
Other heat-shock genes associated with the ribosome are induced under conditions of translational misfolding. Hsp15 (yrfH) binds RNA (Sambrook et al., 1989) and is associated with free 50S ribosomal subunits containing a nascent polypeptide chain (Korber et al., 2000
). Heat shock also increases the level of Hsp15-binding implying increased dissociation of 50S and 30S subunits. Further suggestion of ribosomal dissociation comes from the induction of ftsJ (rrmJ). The ftsJ gene product is an RNA methylase specific for 23S rRNA only when contained in the 50S ribosomal subunit (Caldas et al., 2000a
,b
; Puglisi et al., 2000
). This enzyme methylates 23S rRNA at position 2552 located within the peptidyl transferase center of the ribosome (Caldas et al., 2000a
). Mutants in ftsJ lack methylation of 23S rRNA and show up to 65% decrease in ribosomal activity corresponding to dissociation of the 50S and 30S subunits (Caldas et al., 2000b
). Particularly striking in the rpoH mutant is the large increase in transcripts of the cold-shock proteins (CSPs). Table II
shows the response of CSP transcripts to misfolded protein in the rpoH mutant and the wild-type rpoH strain. These genes are not affected by heat shock (9), but are associated with a transient halt of translation. CSPs are RNA-binding proteins which act as chaperones for untranslated message (Jiang et al., 1997
; Wang et al., 1999
) and provide anti-termination activity (Bae et al., 2000
). Increased expression of CSPs under conditions that reduce chaperone expression (rpoH606) is an indication of paused translation. Taken together, these results suggest a translational regulatory response to misfolded protein. Such regulation might involve rRNA demethylation, as a consequence of translational misfolding. This hypothesis is an interesting regulatory mechanism currently under investigation.
Other induced genes
yccV, yhdN and yrfG have been shown to increase expression under heat-shock conditions but are of unknown function. In addition to these known heat-shock genes, yagU, yciS, ybeD, yejG and yhgI show increased expression. Most of these proteins are relatively small and generally acidic. One speculation is that some of these proteins perform a similar role to IbpAB in the direct recognition and sequestering of misfolded protein. However, only IbpAB have been associated with misfolded and aggregated protein. Induction levels of ibpAB are much higher and these other proteins may be present at lower levels. Interestingly, knockout mutations of ibpAB have relatively little affect on cell growth and viability (Thomas and Baneyx, 1998; data not shown) suggesting some functional redundancy within the cell.
Genetic reporter of protein folding
To confirm the profiling results and facilitate experimentation with a larger number of recombinant proteins, we cloned the promoter regions from ibpAB, ybeD, yhgI and yrfGHI into a ß-galactosidase reporter vector. In each case, increased ß-galactosidase activity was observed when expression of the misfolded protein LCK was induced whereas the folded protein PLA showed no increase in activity. These results were further extended using a set of eight misfolded proteins and six properly folded proteins co-expressed in the presence of the ibpAB-promoter ß-galactosidase fusion. In each case, increased ß-galactosidase activity corresponded to expression of misfolded protein (data not shown). A more detailed characterization is shown below. The response observed, then, appears to be a general result of protein misfolding rather than a specific response to any particular protein. These reporters provide a simple means of identifying misfolded protein through a sensitive enzymatic assay and the ibpAB promoter fusion was chosen as the reporter for further studies.
ELISA-like assay for soluble protein
For identifying protein derivatives that have improved folding properties in a recombinant environment, we also developed an ELISA-like assay compatible with high-throughput screening instrumentation. To evaluate soluble protein levels in a high-throughput system, non-denatured cell lysates must be prepared using conditions compatible with rapid screening in microplates. In lieu of the detergent or organic lysis, we added an antibiotic cocktail to each well to induce lysis. Soluble protein fractions were removed, bound to microtiter plates, and recombinant protein detected via binding of a Ni-HRP conjugate to a 6X-histidine N-terminal fusion. It should be noted that the His-tag may not be uniformly accessible among recombinant proteins. A negative Ni-HRP response, therefore, may not be indicative of an absence of soluble protein, but the protein fold may occlude access to the His-tag. However, we have not observed this to be a common problem. This assay, then, provides a measure of the levels of soluble recombinant protein without the need to run an SDS gel and in a form that is compatible with a HT-screen and the ß-galactosidase assay.
Testing proteins with pre-determined expression characteristics
As part of our effort aimed at cloning, expressing and characterizing the total proteome of T.maritima, we tested the efficacy of the reporter on a set of 18 T.maritima proteins (six soluble, six insoluble and six mixed solubility). To optimize assay parameters, strains were arrayed in 96-well plates and assayed in triplicate at three induction levels (0.02, 0.2 and 2% arabinose) and at four post-induction time points for addition of the lysis-promoting antibiotics (t=0, 30, 60 and 120 min after addition of arabinose.) Figure 2 shows the averaged results for triplicate plates (soluble, insoluble and mixed) for the 0.2% arabinose induction. Both the insoluble and the mixed pools showed >4-fold higher ß-galactosidase activity than the soluble pool. Conversely, the soluble pool showed a >10-fold higher response in the Ni-HRP assay opposed to the insoluble pool. The mixed pool, comprised of proteins expressed approximately equally in both soluble and insoluble fractions, showed Ni-HRP binding approximately half the intensity of the soluble pool. Although either lack of ß-galactosidase or presence of Ni-HRP activity alone could be used as a measure of soluble protein, we chose a ratio of the two activities, a more effective and convenient screen.
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We next applied this screen to a large set of proteins with unknown folding properties. We performed the screen under the optimal conditions noted above on 186 T.maritima proteins not previously characterized for expression. The results of this screen are summarized in Table III. SDSPAGE of eluates from nickel-chelating resin and the dissolved insoluble fractions for each clone was performed along with corresponding ß-galactosidase activity, Ni-HRP response and solubility scores for 186 clones. Based on the results of the gels, 57 clones did not overexpress a visible protein band, 62 clones expressed predominantly soluble protein, 27 expressed predominantly insoluble aggregates and 46 expressed approximately equally in both soluble and insoluble fractions. A comparison of ß-galactosidase activity to the Ni-HRP assay is shown in Figure 3
. Points are categorized by SDS gel analysis of the soluble and insoluble protein fractions. The screen positively identified 54 of 62 (87%) soluble proteins. Seven of the eight remaining proteins that were soluble according to the gels had low Ni-HRP assays, most likely due to inaccessibility of the His-tag in these fusion proteins. Taken alone, the ß-galactosidase activity measurement identified 22 of 27 (81%) insoluble proteins. Those proteins showing partial solubility showed variable solubility scores, suggesting partial folding is inducing ß-galactosidase through the reporter. This assay, then, provides an effective and convenient means of classifying folding characteristics.
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The true utility of this system lies in the ability to identify variants of full-length gene products, either mutants or domains, based on improved properties. For structural and biochemical studies, we tested the ability of this screen to identify soluble fragments of Rep68 (GI: 209617), an adeno-associated virus non-structural protein possessing various activities related to the integration of the viral genome into target DNA. This protein previously had been found to express predominantly as unfolded aggregates in E.coli. We performed both a random approach and a rational approach based on selection of domains with regard to homology. Three domains of Rep68 were selected after an RPS-BLAST search (Altschul et al., 1997) identified an internal domain possessing homology to a parvovirus non-structural protein, NP-1. This information, combined with a KyteDoolittle hydropathy plot (Figure 4
), was used to assign the 5' and 3' cut-offs for each domain. The remaining N-terminal and C-terminal residues comprised the other two domains and did not possess significant homology to any other proteins in the database. Random fragments of Rep68 were also generated for screening by DNase fragmentation.
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Discussion |
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The differentially regulated genes identified provide a valuable opportunity to create novel reporters of the folding state of cellular proteins as a whole and overexpressed, recombinant proteins in particular. Our reporter assay differs from others recently described by not relying on direct coupling of the reporter gene to the target, thereby limiting potential interference by the reporter. The combination of the Ni-HRP and ß-galactosidase assays provides an effective means of assaying soluble recombinant proteins in a high-throughput way. We have extended this system to identify mutants and truncations of single gene products as a strategy to identify soluble domains of otherwise misfolded, aggregated proteins. Using this approach, we have identified soluble fragments of Rep68 and anticipate that this assay will provide a general means of isolating recombinant protein suitable for structure/function work.
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Notes |
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2 To whom correspondence should be addressed. E-mail: lesley{at}gnf.org
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Acknowledgments |
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References |
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Received August 3, 2001; revised October 31, 2001; accepted November 7, 2001.