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recombinant protein folding and misfloding in E.coli


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? 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology

Recombinant protein folding and misfolding in Escherichia coli
Fran?ois Baneyx & Mirna Mujacic
The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding. Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form. The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E. coli will remain an important host for the production of both commodity and high value added proteins.

The enteric bacterium Escherichia coli is one of the most extensively used prokaryotic organisms for genetic manipulations and for the industrial production of proteins of therapeutic or commercial interest. Compared with other established and emerging expression systems1, E. coli offers several advantages, including growth on inexpensive carbon sources, rapid biomass accumulation, amenability to high cell-density fermentations and simple process scale-up. Because of its long history as a model system, E. coli genetics are very well characterized and many tools have been developed for chromosome engineering and to facilitate gene cloning and expression. If heterologous proteins do not require complex post-translational modifications and are expressed in a soluble form, E. coli is usually first selected to obtain enough material for biochemical and/or structural studies and for the subsequent large-scale production of valuable gene products. It is, however, not uncommon that overexpressed recombinant proteins fail to reach a correct conformation and undergo proteolytic degradation or associate with each other to form insoluble aggregates of nonnative proteins known as inclusion bodies. Over the past 20 years, there has been considerable progress in the fundamental understanding of the mechanisms used by E. coli to support de novo protein folding, manage stress-induced protein misfolding and decide whether misfolded polypeptides should be refolded or degraded. Here, we review this body of knowledge and how it has been exploited to promote the high-level production of heterologous proteins in a correct and bioactive conformation in the bacterial cytoplasm and periplasm. Protein misfolding and inclusion body formation In the crowded milieu of the E. coli cytoplasm where transcription and translation are tightly coupled and one protein chain is released from
Departments of Chemical Engineering and Bioengineering, University of Washington, Box 351750, Seattle, Washington 98195, USA. Correspondence should be addressed to F.B. (baneyx@u.washington.edu). Published online 4 November 2004; doi:10.1038/nbt1029

the ribosome every 35 seconds2, an environment where macromolecule concentration can reach 300–400 mg/ml (ref. 3), protein folding is an extraordinary challenge. In general, small (<100 residues), single domain host proteins efficiently reach a native conformation owing to their fast folding kinetics, whereas large multidomain and overexpressed recombinant proteins often require the assistance of folding modulators. Folding helpers include molecular chaperones, which favor on-pathway folding by shielding interactive surfaces from each other and from the solvent, and folding catalysts that accelerate ratelimiting steps, such as the isomerization of peptidyl-prolyl bonds from an abnormal cis to a trans conformation and the formation and reshuffling of disulfide bonds. For a heterologous protein, failure to rapidly reach a native conformation or to interact with folding modulators in a timely fashion has two possible consequences: partial or complete deposition into insoluble aggregates known as inclusion bodies or degradation. The likelihood of misfolding is increased by the routine use of strong promoters and high inducer concentrations that can lead to product yields exceeding 50% of the total cellular protein. Under such conditions, the rate of protein aggregation is often much greater than that of proper folding and folding modulators are rapidly titrated. A second factor contributing to inclusion body formation is the inability of bacteria to support all post-translational modifications that a protein requires to fold. For instance, the formation of intra- or intermolecular disulfides is not possible in the reducing cytoplasm of wild-type E. coli, which results in the aggregation of certain disulfide bond–rich proteins (e.g., Fab antibody fragments). Inclusion bodies can accumulate in the cytoplasm or periplasm depending on whether or not a recombinant protein has been engineered for secretion. The target typically accounts for 80–95% of the inclusion body material and is contaminated by outer membrane proteins, ribosomal components and a small amount of phospholipids and nucleic acids that likely adsorb upon cell lysis4. Folding modulators (e.g., DnaK, GroEL and IbpA/B) are sometimes—but not always—associated with inclusion bodies5,6. Cytoplasmic inclusion

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Table 1 Cytoplasmic chaperones
Family Name Cofactors Function Disaggregase Possible folding/secretory chaperone DnaJ, GrpE HscB YbeV, YbeS GroES Folding chaperone Iron-sulfur cluster protein assembly σ70 regulation Folding chaperone Holding chaperone Holding chaperone Holding chaperone Holding chaperone, PPIase Substrate specificity Segments enriched in aromatic and basic residues Unknown Segments of four to five hydrophobic amino acids, enriched in leucine and flanked by basic residues HscA LPPVK motif in iron-sulfur cluster protein assembly IscU Unknown α/β folds enriched in hydrophobic and basic residues Unknown Unknown Unknown Eight amino acid motif enriched in aromatic and basic residues SecB SecB Secretory chaperone Nine amino acid motif enriched in aromatic and basic residues
aAAA, ATPases associated with a variety of cellular activities. bATP binding negatively regulates the chaperone activity of Hsp31 at high temperatures23. cATP binding to certain small Hsps triggers conformational changes and substrate release17.

ATP requirement + + + + + + – –b –c –

Hsp100 (AAA+)a ClpB Hsp90 Hsp70 HtpG DnaK

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HscC Hsp60 Hsp33 GroEL Hsp33

DJ-1 superfamily Hsp31 Small Hsps PPIase IbpA, IbpB TF



bodies are porous ovoids or cylinders with maximum characteristic length and volume of 1 ?m and 0.6 ?m3, respectively6–8. However, hemispherical inclusion bodies of 0.5-?m diameter have been observed in the periplasm7. In the cytoplasm, inclusion bodies grow from structured folding intermediates9 at nearly constant rates and around nucleation cores that are mutually exclusive. Thus, multiple inclusions of different sizes may be present within a single cell6. Because inclusion bodies are resistant to proteolysis and contain large amounts of relatively pure material, their formation is often exploited for the production of proteins that are toxic, unstable or easy to refold. Finding optimal conditions for efficient refolding requires considerable optimization, but acceptable yields can usually be achieved using established strategies10. Cytoplasmic folding modulators In E. coli and other systems, host protein misfolding is not uncommon. It may result from premature termination of translation, failure of a newly synthesized chain to reach a correct conformation or from loss of structure triggered by environmental stress. To cope with this situation, cells have evolved largely conserved mechanisms to favor proper de novo folding, refold partially folded proteins, dissolve aggregates and dispose of irretrievably damaged proteins. Molecular chaperones, a ubiquitous class of folding modulators, play a central role in the conformational quality control of the proteome by interacting with, stabilizing and remodeling a wide range of nonnative polypeptides. Although constitutively expressed under balanced growth conditions, many chaperones are upregulated upon heat shock or other insults that increase cellular protein misfolding (including heterologous protein expression11) and are therefore classified as stress or heat shock proteins (Hsps). Mechanistically, molecular chaperones rely on the differential exposure of structured hydrophobic domains to the solvent to bind nonpolar segments that would normally be buried within the core of their substrates. Although there are subtle differences in the composition of client protein recognition sequences (and thus some degree of selectivity in substrate capture), the typical chaperone target is a short unstructured stretch of hydrophobic amino acids flanked by basic residues and lacking acidic

residues (Table 1). The fact that such motifs are common explains why chaperones are so promiscuous. Molecular chaperones can be divided into three functional subclasses based on their mechanism of action (Fig. 1). ‘Folding’ chaperones (e.g., DnaK and GroEL) rely on ATP-driven conformational changes to mediate the net refolding/unfolding of their substrates. ‘Holding’ chaperones (e.g., IbpB) maintain partially folded proteins on their surface to await availability of folding chaperones upon stress abatement. Finally, the ‘disaggregating’ chaperone ClpB promotes the solubilization of proteins that have become aggregated as a result of stress. In the E. coli cytoplasm, de novo folding involves three chaperone systems: trigger factor (TF), DnaK-DnaJ-GrpE and GroEL-GroES (reviewed in refs. 12,13). TF, a three-domain protein that binds ribosomes with moderate affinity in the vicinity of the peptide exit site, is ideally positioned to interact with short nascent chains. Although the central domain of TF exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity, proline residues are not necessary for substrate capture14 and native TF clients are primarily large (>60 kDa) multidomain proteins, which represent 10–20% of the E. coli proteome15. Longer nascent chains or newly synthesized proteins may alternatively be captured by DnaK, a chaperone whose substrate pool overlaps with that of TF15. DnaK is targeted to high-affinity sites by the cochaperone DnaJ, which activates tight substrate binding by triggering hydrolysis of DnaK-bound ATP. Substrate ejection is controlled by GrpE-catalyzed ADP/ATP exchange. Once released, a newly synthesized protein may reach a native conformation, undergo additional cycles of interactions with DnaK (and possibly TF) until it folds, or be transferred to the downstream GroEL-GroES system which handles about 10% of newly synthesized host proteins16. GroEL is an ≈800-kDa oligomer organized as two stacked homoheptameric rings, one of which is always bound by the cochaperone GroES12. GroEL substrates, which consist of structured but nonnative proteins up to 60-kDa in size, are bound by the free ring and allowed to fold at infinite dilution within the central chamber in a process controlled by reversible GroES capping and conformational changes orchestrated by ATP binding and hydrolysis12.

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Figure 1 Chaperone-assisted protein folding in the cytoplasm of E. coli. Nascent polypeptides requiring the assistance of molecular chaperones first encounter TF or DnaK-DnaJ. Both chaperones engage solvent-exposed stretches of hydrophobic amino acids, shielding them from the solvent and each other. After undocking from TF- or GrpE-mediated release from DnaK, folding intermediate may reach a native conformation, cycle back to DnaK-DnaJ or be transferred to the central chamber of GroEL for folding at infinite dilution upon GroES capping. In times of stress (red arrows), thermolabile proteins unfold and aggregate. IbpB binds partially folded proteins on its surface to serve as a reservoir of unfolded intermediates until folding chaperones become available and intercalates within large aggregates. The holding chaperones Hsp33 and Hsp31 become important under oxidative and severe thermal stress, respectively. ClpB promotes the shearing and disaggregation of thermally unfolded host proteins and cooperates with DnaK-DnaJ-GrpE to reactivate them once stress has abated. Recombinant proteins that miss an early interaction with TF or DnaK/DnaJ, that undergo multiple cycles of abortive interactions with folding chaperones or titrate them out, accumulate in inclusion bodies (green arrows).

J K TF 3′ ′ 5′ ′
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ClpB Native ADP
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In addition to mediating proper de novo folding, DnaK and GroEL refold host proteins that become unfolded when cells experience environmental stress. They are assisted in this task by holding chaperones (holdases) that stabilize partially folded proteins without actively promoting their remodeling (Fig. 1 and Table 1). The most extensively characterized holdases belong to the small Hsp family17. The bacterial representatives, IbpA and IbpB, are two homologous 16-kDa proteins encoded on a single operon5. IbpB forms large oligomers and relies on temperature-driven exposure of structured hydrophobic domains to capture unfolded intermediates of denaturation-prone proteins on its surface18. Once stress abates, IbpB-bound species are engaged by DnaK, and if necessary transferred to GroEL, for refolding19. Hsp33, which was identified on the basis of its thermal induction, is also classified as a holdase20. The main function of this redoxregulated chaperone is to manage oxidative protein misfolding20. Under balanced conditions, Hsp33 is a reduced monomer that coordinates a zinc atom via four conserved cysteines. When cells are exposed to reactive oxygen species—a situation that often accompanies heat shock—the cytoplasm becomes more oxidizing, Hsp33 monomers form intramolecular disulfide bonds, which trigger zinc release, and the protein adopts a dimeric conformation exhibiting chaperone activity20. The thioredoxin and glutaredoxin systems (see below) rapidly reduce Hsp33 disulfides in a process that does not cause substrate release but primes the chaperone for fast inactivation. Upon return to nonstress conditions, DnaK-DnaJ engage the bound substrate and refold it alone or with the help of GroEL-GroES21. Hsp31, a recently characterized cytoplasmic folding modulator22,23 also functions as a holdase that binds early unfolding intermediates in times of severe stress, thereby preventing overloading of the DnaKDnaJ-GrpE system24. The interface of this homodimer of 31-kDa units contains a ≈20-? hydrophobic bowl proximal to flexible linker-loop regions that shield large nonpolar patches on either side of the bowl25,26. Temperature-induced motion of the linker-loop domains

allows efficient capture of unfolding intermediates by uncovering high-affinity binding sites adjacent to the bowl27. The linker-loop region may also play a role in substrate ejection by returning to its original position upon stress abatement. Although Hsp31 is not an ATPase, its chaperone activity is negatively regulated by ATP binding at high temperature23, possibly to coordinate substrate capture with the needs of the chaperone network. Because folding and holding chaperones fail to abrogate protein aggregation under severe or prolonged stress conditions, E. coli possesses a third line of defense to manage the deleterious effects associated with misfolding: active aggregate solubilization. Disaggregation is performed by ClpB, a member of the Hsp100 family of ring-forming ATPases that also include ClpA, ClpX and ClpY, three proteins whose primary function is in proteolysis28 (see below). The structure of Thermus thermophilus ClpB suggests that solubilization may rely on both a ‘crowbar’ action involving a long surface exposed coiled-coil domain, as well as net unfolding of the substrate by threading through the ≈16-? central pore of the chaperone29,30. ClpB-mediated disaggregation is facilitated by intercalation of small Hsps within the aggregates31 but renaturation requires transfer of partially folded substrate from ClpB to DnaK32,33. Interestingly, DnaK-DnaJ can solubilize small aggregates in vitro34 and their interaction with large aggregates may be necessary for the initial steps of ClpB-driven disaggregation35. More poorly characterized folding modulators include HtpG, which may play a role in de novo folding and secretion, the specialized DnaK paralogs HscA and HscC (Table 1), and SlpA and SlyD, two homologous PPIases of unclear function. Protein export Proteins synthesized in the cytoplasm may remain in this compartment, integrate within the inner membrane or translocate to the periplasm36 (Fig. 2). In E. coli, the vast majority of proteins destined for export are secreted by the Sec-dependent pathway and are

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DegP Prc

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? 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology

SH SH SH

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Figure 2 Export and periplasmic folding pathways. Proteins destined for export can be translocated across the inner membrane in three different fashions. (a) Preproteins with highly nonpolar signal sequences (green) or transmembrane segments of inner membrane proteins are recognized by SRP which, along with TF, scans nascent chains. SRP-dependent export involves delivery of the ribosome-nascent chain complex to FtsY and subsequent translocation through the SecYEG-SecDFYajC translocon. (b) The vast majority of preproteins have less hydrophobic signal sequence (lavender) and undergo Sec-dependent export. TF associates with the nascent polypeptide, halting cotranslational folding. As the chain grows, TF dissociates and the polypeptide is transferred to SecB or DnaK that maintain it in an extended conformation. Delivery to SecA and ATP-dependent translocation through SecYEG completes the process. (c) Preproteins with signal sequences containing the twin-arginine motif (cyan) are exported via the Tat-dependent pathway in a folded form. After cleavage of the signal sequence, partially folded periplasmic proteins may aggregate (1), undergo proteolysis (2) or reach a native conformation, possibly with the assistance of folding modulators (3). Cysteine pairs in proteins containing disulfide bonds are oxidized by DsbA (4) whereas incorrect disulfides are isomerized by DsbC (5). These oxidoreductases are reactivated by DsbB and DsbD, respectively. Black arrows show products obtained after each step, whereas blue arrows represent electron flow.

synthesized with an amino-terminal signal sequence, 20–30 amino acids in length, that consists of a hydrophobic core followed by a proteolytic cleavage site. Efficient export of the resulting preproteins requires targeting to the membrane-associated translocation apparatus in an extended conformation. The homotetrameric secretory chaperone SecB maintains large (>200 residues) preproteins in an export-competent form by using two 70-?-long hydrophobic channels running along its sides37. Although generic chaperone such as DnaK and GroEL can also perform this duty38, SecB has the advantage of containing an acidic ‘top’ region that allows it to dock and transfer the protein cargo to the peripheral membrane protein SecA37. Through cycles of ATP-hydrolysis, SecA drives itself and the preprotein into the pore formed by the integral membrane proteins SecYEG. Translocation to the periplasm is dependent upon the proton motive force and facilitated by the SecDFYajC complex (Fig. 2, path a). In the process, the signal sequence is removed by the membrane-associated Lep or Lsp signal peptidases (the latter being specific for glyceridemodified prolipoproteins). A subset of proteins is exported via the signal recognition particle (SRP)-dependent pathway39 (Fig. 2, path b). Bacterial SRP, composed

of a 48-kDa GTPase termed Ffh and the 114 nt-long 4.5S RNA, can bind either to the signal sequence of certain secretory proteins (provided that it is highly hydrophobic) or to transmembrane segments of inner membrane proteins as they emerge from the ribosome. The SRP-bound ribosome nascent chain complex (RNC) is then targeted to the membrane-bound receptor FtsY. Upon GTP hydrolysis by SRP and its receptor, the SRP-RNC-FtsY complex dissociates and the RNC is transferred to SecYEG for cotranslational translocation in a process that involves SecA40. Because of its ability to bind nascent proteins, TF also plays a role in both Sec- and SRP-dependent protein secretion. In the former pathway, TF seems to sequester nascent chains for relatively long periods of times (perhaps until half of a typical 30-kDa protein has been translated), thereby allowing larger, unstructured proteins to be efficiently engaged by SecB41. In the case of SRP-dependent export, TF and SRP, which share a common attachment site (the L23 ribosomal protein), both sample nascent chains exiting from the ribosome. The appearance of a highly hydrophobic signal sequence or transmembrane segment leads to high-affinity SRP binding, and subsequent interaction with FtsY results in TF ejection and initiation of cotranslational translocation41.

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maturation of trimeric outer membrane proteins52. It resembles an asymmetric dumbbell and contains a deep cleft within its core modClassification Protein Substrates ule that may be responsible for substrate Generic chaperones Skp (OmpH) Outer membrane proteins and misfolded binding53. The fact that SurA preferentially periplasmic proteins recognizes an Ar-X-Ar motif (where Ar is an FkpA Broad substrate range aromatic and X any residue) that is common Specialized chaperones SurA Outer membrane proteins in outer membrane proteins but infrequent in LolA Outer membrane lipoproteins other polypeptides may explain its substrate PapD (and its family) Proteins involved in P pili biosynthesis specificity54. FimC Proteins involved in type 1 pili biosynthesis One of the features that distinguish the periplasm from the cytoplasm is its oxidizing PPIases SurA Outer membrane β-barrel proteins environment. Indeed, in wild-type E. coli, staPpiD Outer membrane β-barrel proteins bly disulfide-bonded proteins are only found FkpA Broad substrate range PpiA (RotA) Unknown in the cell envelope where disulfide formation and isomerization is catalyzed by a set of Proteins involved in DsbA Reduced cell-envelope proteins thiol-disulfide oxidoreductases known as the disulfide bond formation DsbB Reduced DsbA Dsb proteins55,56. DsbA, a soluble periplasmic DsbC Proteins with nonnative disulfides protein containing a C-P-H-G active site DsbG Proteins with nonnative disulfides embedded in a thioredoxin-like fold uses its DsbD Oxidized DsbC, DsbG and CcmG highly reactive Cys30 to promote disulfide DsbE (CcmG) Cytochrome c biogenesis transfer to substrate proteins by the formaCcmH Cytochrome c biogenesis tion of mixed disulfide species (Fig. 2, path 4). It is kept in an active oxidized state by DsbB, an inner membrane protein exposing two Whereas both Sec- and SRP-dependent pathways handle pre- loops to the periplasm, each containing two cysteines. DsbA recycling proteins that have not yet reached a native conformation, the twin- involves initial attack of the Cys104-Cys130 DsbB disulfide and coorarginine (Tat)-dependent secretion pathway exclusively deals with dinated involvement of all four DsbB cysteines before release of oxifolded or partially folded proteins. Proteins exported via the Tat path- dized DsbA56. If incorrect disulfide bonds form in proteins containing way are produced with a signal sequence that contains a conserved— more than two cysteines, the disulfide bond isomerase DsbC comes to but not absolutely required42—twin arginine motif and most natural the rescue (Fig. 2, path 5). This soluble V-shaped homodimer is strucsubstrates are redox cofactor-binding proteins necessary for anaerobic turally similar to Skp with N-terminal dimerization domains and respiration. Four integral membrane proteins, TatA, TatB, TatC and C-terminal thioredoxin folds containing C-G-Y-C active sites57. DsbC TatE, make up the Tat-export machinery (Fig. 2, path c). Although, the is thought to capture folding intermediates within the uncharged cleft precise mechanism of Tat-dependent transport remains controver- formed by its dimerization interface and to use its reduced Cys98 to sial43, it has been postulated that the TatBC complex recognizes sub- attack disulfides in substrate proteins, thereby catalyzing isomerizastrate proteins and delivers them to TatA, which forms a transport tion in a process involving mixed disulfide intermediates. DsbC is channel capable of accommodating substrates with diameters of up to maintained in a reduced state by the inner membrane protein DsbD at the expense of NADH oxidation in the cytoplasm55,56. DsbA, DsbC 70 ?44. TatE seems to be interchangeable with TatA45. and DsbG all exhibit chaperone activity, presumably because a parPeriplasmic folding modulators tially folded structure is needed to allow efficient disulfide formation The periplasm contains a single bona fide chaperone termed Skp that and isomerization in substrate proteins. captures unfolded proteins as they emerge from the Sec translocation apparatus (Fig. 2) and whose primary function is to assist the folding Proteolysis and membrane insertion of outer membrane proteins46,47. In the Skp The degradation of misfolded proteins by host proteases guarantees homotrimer, α-helical tentacles extending from a β-barrel body define that abnormal polypeptides do not accumulate within the cell and a central cavity that can accommodate nonnative substrates or folding allows amino acid recycling. Targets for degradation include premamodules up to ≈20-kDa in size48. Consistent with the absence of a turely terminated polypeptides, proteolytically vulnerable folding periplasmic ATP pool, Skp chaperone activity is ATP independent48 intermediates that are kinetically trapped off-pathway, and partially folded proteins that have failed to reach a native conformation after and this chaperone is likely a holdase. Other periplasmic folding modulators include the PPIases SurA, multiple cycles of interactions with folding modulators. In the cytoplasm, proteolytic degradation is initiated by five ATPFkpA, PpiA and PpiD (Table 2). Among these, FkpA has the most generic folding activity49 and combines PPIase and chaperone func- dependent heat shock proteases (Lon, ClpYQ/HslUV, ClpAP, ClpXP tions50. FkpA is a V-shaped homodimer with N-terminal segments and FtsH) and completed by peptidases that hydrolyze sequences 2–5 responsible for dimerization and chaperone activity and C-terminal residues in length. These proteases consist of a remodeling component PPIase domains51. FkpA is believed to cradle partially folded sub- or domain that binds substrate proteins and couples ATP hydrolysis to strates within the hydrophobic cleft formed at the dimerization inter- unfolding and transfer of the polypeptide to an associated protease face, allowing the flexible C-terminal domains easy access to prolyl domain or proteolytic component. Lon is a tetrameric serine protease of 87-kDa subunits containing bonds requiring isomerization. SurA, which contains two parvulin-like PPIase domains, relies on three functional domains. Its N-terminus is involved in substrate its chaperone activity–rather than PPIase activity–to support the recognition and binding whereas its central and C-terminus domain
Table 2 Periplasmic folding modulators

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are responsible for ATPase and proteolytic activities, respectively. In addition to being responsible for bulk protein degradation58,59, Lon also exerts a regulatory function by degrading a class of proteins that are designed to be unstable (e.g., SulA). ClpA (84 kDa), ClpX (46 kDa) and ClpY (49 kDa) assemble into hexameric ATPase rings that recognize and actively unfold proteins destined for degradation or remodeling by threading them through a central channel in a process fueled by ATP-driven conformational changes60. Both ClpA and ClpX associate with the same serine proteolytic component ClpP (a protein organized as two stacked heptamers of 23-kDa subunits) via ‘ClpP loops’ containing a [L/I/V]-G-[F/L] tripeptide absent in ClpB61. Because ClpP presents two identical faces for ClpA/ClpX interactions, complexes consisting of one or two ClpA or ClpX rings bound to one ClpP double-ring as well as heteromeric ClpA:ClpP:ClpX complexes can all form in vitro62. It has been suggested that ClpA:ClpP:ClpA particles may be best suited to carry out the remodeling of a subset of substrate proteins that are released without degradation62. ClpY specifically binds to either or both ends of ClpQ, a proteolytic component organized as two stacked hexameric rings of 19-kDa subunits, to form a structure that resembles the eukaryotic 26S proteaseome63. Together with Lon, ClpYQ is thought to be primarily responsible for the degradation of abnormal proteins58. Although also involved in bulk proteolysis, ClpXP, and to a lesser extent ClpAP, specifically degrade prematurely terminated proteins that have been modified by attachment of an SsrA tag (AANDENYALAA) at their C terminus64. Shuttling of tagged substrates to ClpX requires binding of the adaptor protein SspB to the SsrA tag and its subsequent interaction with the N terminus of ClpX65. ClpAP does not appear to require such an usher protein. However, an adaptor protein termed ClpS that associate with the N terminus of ClpA redirects ClpAP protease activity from soluble proteins to aggregated species66. FtsH (HflB), the only ATP-dependent cytoplasmic protease associated with the inner membrane, is organized as an hexamer of 71-kDa subunits that associates with dimers or hexamers of the HflK-HflC inner membrane proteins to form 1-MDa complexes67. FtsH relies on its cytoplasmic metalloprotease active site to degrade both membraneembedded and soluble cytosolic proteins including the heat shock sigma factor σ32 and SsrA-tagged proteins. The role of HflKC in FtsH function is poorly understood. Accumulation of misfolded proteins also occurs in the cell envelope owing to temperature increase, oxidative stress or improper formation of disulfide bonds. The primary housekeeping periplasmic protease is DegP a hexamer formed by staggered association of trimeric rings68. The proteolytic sites of DegP are located within an inner cavity bounded by mobile side walls formed by PDZ domains. PDZ regions control access to the protease chamber and are likely involved in substrate binding69. At low temperatures, DegP switches function from protease to chaperone70 but the physiological relevance of this activity remains unclear. A second generic periplasmic protease, Prc (Tsp), cleaves proteins with nonpolar C-termini, membrane proteins and unfolded polypeptides with broad primary sequence specificity71. Prc contains a single PDZ domain that has been implicated in substrate binding72. Additional cell envelope proteases include DegS, DegQ, Protease III and OmpT. DegS is an inner membrane homotrimer that senses protein misfolding by binding Y-X-F tripeptides (where X is any amino acid) exposed at the C-terminus of immature outer membrane porins. Motion of the PDZ domains results in the cleavage of RseA, a transmembrane protein that sequesters the extracytoplasmic factor σ24, thereby allowing high-level production of proteins involved in combating protein misfolding in the cell envelope73. The more poorly characterized serine protease DegQ hydrolyzes denatured substrates at discrete V/I-X locations74, whereas protease III (the ptr gene product) is involved in the degradation of both protein fragments and larger abnormal proteins75. OmpT is an outer membrane protein specific for paired basic residues, which is organized in a vase-like structure with a serine active site that faces the growth milieu76. Because OmpT readily adsorbs to inclusion bodies during cell lysis and remains active under highly denaturing conditions77, ompT null strains should always be used if the refolding route is chosen. Cytoplasmic folding pathways engineering Many eukaryotic proteins of therapeutic or commercial interest possess complex tertiary and quaternary structures and often require the formation of multiple disulfide bonds and other post-translational modifications to reach a native, biologically active conformation. Producing these proteins in E. coli can be challenging because the cellular environment, folding machinery and conformational quality control checkpoints of prokaryotes are quite different from those of eukaryotes. Not surprisingly, inclusion body formation and proteolytic degradation are commonly observed upon heterologous protein overexpression in E. coli. A traditional approach to alleviate these problems involves reducing the synthesis rate of the target gene product to promote proper folding. This can be achieved by using weaker promoters or by decreasing the concentration of gratuitous inducer. For promoters based on lac-derived control elements (e.g., the tac or trc promoters), isopropyl D-thiogalactopyranoside (IPTG) concentrations below 100 ?M are suitable for partial induction. However, because the PBAD promoter operates in an ‘all or none’ fashion in wild-type cells, graded induction by subsaturating arabinose concentrations is only possible in strains that have been engineered to constitutively transport arabinose78,79. An alternative strategy is to decrease the temperature at which the recombinant protein accumulates. The use of low temperatures has the combined advantages of slowing down transcription and translation rates and of reducing the strength of hydrophobic interactions that contribute to protein misfolding. The drawback of this approach (as is the case with low inducer concentrations) is a reduction in productivity. Because traditional promoter systems exhibit reduced efficiency below 15°C, cold-inducible promoters, such as that of the major E. coli cold-shock gene cspA, are best suited for driving transcription at very low temperatures80. cspA-driven transcription is also useful for the expression of proteolytically sensitive and membraneassociated gene products81 and companion strains that relieve promoter repression after prolonged incubation at low temperature are available82. Second generation cspA-based expression vectors have recently been described83. One of the most extensively used approaches to improve the yields of soluble proteins in the E. coli cytoplasm involves coexpression of molecular chaperones implicated in de novo protein folding. The beneficial effects of an increase in the intracellular concentration of TF, DnaK-DnaJ (with or without GrpE) and GroEL-GroES is well documented and a number of plasmids compatible with the routinely used ColE1-derived expression vectors are available84. DnaK-DnaJ or TF overexpression is suitable to increase the solubility of proteins requiring the assistance of chaperones in the early stages of their folding pathway85. For folding intermediates that rapidly transit through the TF/DnaK or require help at later folding stages, GroEL-GroES coexpression may be most beneficial. Technically, the GroEL-GroES encapsulation mechanism should limit the usefulness of this system to proteins smaller than ≈60 kDa. Nevertheless, larger proteins may also

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benefit from GroEL-GroES coexpression86, presumably because GroES-independent stabilization of partially folded domains by GroEL facilitates correct folding of the remainder of the chain87. If aggregation-prone intermediates are formed at both early and late stages of the folding pathway, coordinated expression of DnaK-DnaJ (or TF) and GroEL-GroES may be required to maximize recovery of the target protein in a soluble form85,88. Nevertheless, there are many—and often unpublished—studies in which coexpression of folding modulators fails to improve recombinant protein solubility. The underlying mechanisms are unclear but may be related to the need for timely interactions with specific folding modulators or to the substrate folding pathway itself. Indeed, it was recently reported that binding of TF and DnaK to nascent firefly luciferase chains redirects the folding of this protein from an efficient eukaryotic cotranslational mode to a slower post-translational pathway that is accompanied by aggregation89. Consequently, certain multidomain eukaryotic proteins that have evolved to take advantage of cotranslational folding may not benefit from chaperone overproduction. It should finally be noted that chaperone overexpression may also reduce the overall yield of recombinant proteins90 (defined as the sum of soluble and insoluble fractions), possibly by transiently stabilizing off-pathway intermediates that are subsequently degraded by host proteases. In such cases, an increase in the yields of soluble and bioactive product may be achieved in strains bearing mutations in chaperone systems91. Stable disulfide bonds do not form in the cytoplasm of E. coli owing to their rapid reduction by the combined action of thioredoxins and glutaredoxins92. Enzymes from both pathways share a thioredoxin fold and a C-X-X-C active site. In their reduced form, the thioredoxins TrxA and TrxC attack disulfides in substrate proteins and leave them reduced while becoming oxidized in the process. Thioredoxin reductase (TrxB) recycles oxidized TrxA/C by reducing active site disulfides in an NADPH-dependent manner. GrxA, GrxB and GrxC perform a similar disulfide bond–reductase function but belong to the glutaredoxin pathway. They are kept in a reduced state through the action of tripeptide glutathione (the product of the gshA and gshB genes), which is in turn reduced by glutathione reductase (Gor). Identification of the members of thioredoxin and glutaredoxin pathways and subsequent elucidation of their roles in disulfide bond reduction has made it possible to manipulate the E. coli cytoplasm to rationally promote disulfide bond formation in heterologous proteins. Production of oxidized proteins in the cytoplasm was first demonstrated in trxB mutants93 and later shown to be due to a reversal of function of TrxA and TrxC from reductases to oxidases, owing to their accumulation in a disulfide-bonded form in the absence of TrxB94. Later work showed that the cytoplasm could be made even more reducing by incubating trxB cells at low temperatures90,95 or by combining trxB and gshA or gor null mutations96. Although aerobic growth of the double mutants was impeded96, suppressor strains exhibiting good growth characteristics were isolated and shown to be suitable for enhancing disulfide bond formation in heterologous proteins97,98. The yields of properly disulfide-bonded proteins in trxB gor suppressor cells can be further increased by coexpressing folding modulators including TF, GroEL-GroES and variants of Skp and DsbC that remain cytoplasmic due to the removal of their signal sequences97–99. In the last case, the chaperone activity of DsbC, rather than its disulfide isomerase activity, is determining in enhancing folding98. Despite the usefulness of the strategies described in the above paragraphs, recombinant gene products are commonly sensitive to proteolysis. In such cases, strains lacking ATP-dependent cytoplasmic proteases may be useful expression hosts, particularly if a single protease is responsible for degradation and if production is carried out at the laboratory scale. However, this approach is not without drawbacks. For example, thermosensitive ftsH mutants exhibit poor growth characteristics and E. coli K-12 mutants lacking Lon are filamentous and unsuitable for high-density fermentations. The fact that the E. coli B strain BL21 retains good growth characteristics while lacking both lon and ompT explains the popularity of this host. It is, however, not known if the cells compensate for these deficiencies by upregulating the concentration of other proteases. Export pathways engineering Recombinant proteins can be targeted to the periplasmic space in a Sec-dependent fashion by fusing naturally occurring signal sequences (e.g., those of PelB, OmpA, MalE or PhoA) to their N terminus. Periplasmic expression has a number of advantages over cytoplasmic production. First, an authentic N terminus can be obtained after removal of the signal sequence by leader peptidases. Second, the periplasm is conducive to disulfide bond formation because of the presence of the Dsb machinery. Third, there are fewer proteases in the periplasm compared to the cytoplasm and many have specific substrates. Finally, because the periplasm contains fewer proteins and because its content can be selectively released by osmotic shock or other strategies100,101 purification of the target protein is facilitated. One of the difficulties associated with heterologous protein secretion is inefficient export, which manifests itself by the degradation or aggregation of preproteins in the cytoplasm, and, in the cases of highly hydrophobic or integral membrane proteins, by membrane jamming which is associated with toxicity and eventual cell death. The use of low temperatures81 or the cooverexpression of chaperones involved in secretion (e.g., SecB, DnaK-DnaJ and GroEL-GroES) may alleviate these problems. However, the benefits of the latter approach are highly dependent on the signal-sequence/mature-protein combination102. E. coli mutants originally selected for their ability to support the export of proteins with defective signal sequences offer an alternative route to promote secretion. One such allele, prlA4, encodes a defective version of SecY that leads to enhanced translocation rates, increased affinity of SecA for the SecYEG translocon, reduced reliance of Sec transport on the proton motive force and export of preproteins with folded domains103,104. The observation that inactivation of TF (encoded by tig) accelerates protein export and reduces the dependency of preproteins on secretory factors such as SecB105,106 suggests that ?tig strains will also be useful to enhance heterologous protein secretion. Finally, an artificial increase in signal sequence hydrophobicity may redirect translocation from the Sec- to the SRP-dependent pathway and concomitantly eliminate toxicity effects associated with membrane jamming by tightly coupling secretion with translation105. Owing to its relatively recent characterization, the potential of the bacterial Tat-dependent export pathway for heterologous protein secretion has not yet been fully explored. However, successful translocation of several heterologous proteins (including single chain and Fab antibody fragments107) suggest that Tat-dependent secretion will be a valuable tool for the secretion of heterologous proteins that assume a folded or a partially folded form before reaching the Sec machinery. Already, it has been shown that coexpression of the phage shock protein PspA improves the secretory capacity of the Tat system108. Engineering of periplasmic folding pathways As they emerge in an unfolded or partially folded form on the periplasmic side of the inner membrane, heterologous proteins also confront the task of reaching a native conformation. As previously mentioned, the two cell envelope chaperones exhibiting the widest

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substrate specificity are Skp and the PPIase FkpA (Table 2). Several studies have shown that coexpression of these folding helpers enhances heterologous protein folding and reduces degradation and periplasmic inclusion body formation49,50,109–111. Similarly, cooverproduction of DsbA or DsbC alone or in combination with DsbB and DsbD can facilitate the folding of proteins containing complex patterns of disulfide bonds112–116. It should finally be noted that, because the outer membrane is permeable to small solutes (<600 Da), an alternative way to reduce aggregation in the periplasm is to supplement the growth medium with small nonmetabolizable sugars, such as sucrose and rafinose117,118. By equilibrating within the periplasm, these sugars directly affect folding pathways, presumably by increasing protein chemical potentials via preferential exclusion effects117. The increased understanding of the specificity and mode of action of cell-envelope proteases suggests that a few simple precautions may go a long way in alleviating the problem of periplasmic degradation. First, because DegP is the major housekeeping protease and recognizes commonly occurring paired hydrophobic residues69, degP null hosts should be routinely used. Second, proteins containing nonpolar C-terminal sequence should be expressed in prc (or perhaps degS) mutants, or as N-terminal fusions to carrier proteins. Finally, if proteolysis remains a problem, strains containing multiple mutations in the degP, ompT, ptr and prc genes may prove valuable. Nevertheless, the tradeoff associated with their reduced growth rates should be carefully considered75,119. The road ahead The growing understanding of the principles that govern protein folding and misfolding in E. coli, the availability of sophisticated tools for chromosome engineering and rapid progress in PCR and directed evolution methodologies, have opened many new avenues of research. Already, it is obvious that one is not limited to traditional systems because the chaperone activity either of periplasmic folding helpers (e.g., Skp and DsbC98,99) or of oxidoreductases (e.g., TrxA120,121) can be co-opted to improve the folding of cytoplasmic recombinant proteins. Progress in the study of the structure-function relationship of folding modulators will undoubtedly allow rational improvements and/or coordination of chaperone and catalytic activities to promote more efficient heterologous protein folding. The ‘irrational’ route also holds enormous potential. In a landmark study, Weissman and coworkers122 showed that successive rounds of in vivo screening and DNA shuffling can be used to evolve GroEL variants exhibiting greatly enhanced ability to fold green fluorescent protein. Similar strategies could be applied to other chaperonesubstrate pairs to isolate designer folding modulators dedicated to the efficient folding of specific, high-value recombinant proteins. That ClpB possesses disaggregation activity raises the possibility that engineered or evolved variants could solubilize cytoplasmic inclusion bodies and release component proteins in a conformation committed to folding, thereby enhancing the overall yields of native species during fermentation. Finally, it has recently become apparent that E. coli can be manipulated to achieve post-translational modifications that have long been considered to be beyond its reach. For instance, engineering of the Campylobacter jejuni glycosylation pathway into E. coli has allowed (non-eukaryotic) N-linked glycosylation of the model protein AcrA123. More recently, Schultz and coworkers124 evolved an orthogonal synthetase-tRNA pair suitable for the insertion of an exogenously added glycosylated amino acid (N-acetylglucosamine-serine) in response to amber codons, and obtained good yields of recombinant myoglobin containing N-acetylglucosamine-serine. Because N-acetylglucosamine serves as a substrate for the synthesis of more complex carbohydrates, further strain engineering to coexpress glycosyltransferases may provide an economically viable route for the production of therapeutic glycoproteins. Clearly, progress on the above issues and unexpected new discoveries all but guarantee the future of E. coli as an expression host.
ACKNOWLEDGMENTS This work was supported by National Science Fund award BES-0097430. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturebiotechnology/
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? 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology

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