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EP1451367A2 - Genie de peptides leader pour la secretion de proteines recombinees dans des bacteries - Google Patents

Genie de peptides leader pour la secretion de proteines recombinees dans des bacteries

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Publication number
EP1451367A2
EP1451367A2 EP02795597A EP02795597A EP1451367A2 EP 1451367 A2 EP1451367 A2 EP 1451367A2 EP 02795597 A EP02795597 A EP 02795597A EP 02795597 A EP02795597 A EP 02795597A EP 1451367 A2 EP1451367 A2 EP 1451367A2
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EP
European Patent Office
Prior art keywords
protein
export
bacteria
reporter protein
leader peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02795597A
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German (de)
English (en)
Other versions
EP1451367A4 (fr
Inventor
George Georgiou
Matthew Delisa
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Research Development Foundation
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Research Development Foundation
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Application filed by Research Development Foundation filed Critical Research Development Foundation
Publication of EP1451367A2 publication Critical patent/EP1451367A2/fr
Publication of EP1451367A4 publication Critical patent/EP1451367A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1051—Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/034—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the present invention relates generally to the fields of genetic engineering and protein secretion. More specifically, the present invention relates to engineering of leader peptides for the secretion of recombinant proteins in bacteria.
  • Proteins destined for secretion from the cytoplasm are synthesized with an N- terminal peptide extension of generally between 15-30 amino acids known as the leader peptide.
  • the leader peptide is proteolytically removed from the mature protein either concomitant to or immediately following export into an exocytoplasmic location.
  • polypeptides cross the membrane via a 'threading' mechanism, i.e., the unfolded polypeptides insert into a pore-like structure formed by the proteins SecY, SecE and SecG and are pulled across the membrane via a process that requires the hydrolysis of ATP (Schatz and Dobberstein, 1996).
  • proteins exported through the Tat-pathway transverse the membrane in a partially or perhaps even fully folded conformation.
  • the bacterial Tat system is closely
  • the natural substrates for this pathway are proteins that have to fold in the cytoplasm in order to acquire a range of cofactors such as FeS centers or molybdopterin.
  • proteins that do not contain cofactors but fold too rapidly or too tightly to be exported via any other pathway can be secreted from the cytoplasm by fusing them to a Tat-specific leader peptide (Berks, 1996; Berks et al, 2000).
  • TatB or C completely abolishes export (Bogsch et al, 1998; Sargent et al, 1998; Weiner et al, 1998).
  • the Tat complex purified from solubilized E. coli membranes contained only TatABC (Bolhuis et al, 2001).
  • In vitro reconstitution of the translocation complex demonstrated a minimal requirement for TatABC and an intact membrane potential (Yahr and Wickner, 2001).
  • the choice of the leader peptides, and thus the pathway employed in the export of a particular protein, can determine whether correctly folded functional protein will be produced (Bowden and Georgiou, 1990; Thomas et al, 2001). Feilmeier et al.
  • Tat-GFP propeptide was first able to fold in the cytoplasm and then be exported into the periplasmic space as a completely folded protein (Santini et al, 2001; Thomas et al, 2001).
  • leader peptides other than TorA can be employed to export heterologous proteins into the periplasmic space of E. coli. The cellular compartment where protein folding takes place can have a dramatic effect on the yield of biological active protein.
  • the bacterial cytoplasm contains a large number of protein folding accessory factors, such as chaperones whose function and ability to facilitate folding of newly synthesized polypeptides is controlled by ATP hydrolysis.
  • the bacterial periplasm contains relatively few chaperones and there is no evidence that ATP is present in that compartment.
  • many proteins are unable to fold in the periplasm and can reach their native state only within the cytoplasmic milieu.
  • the only known way to enable the secretion of folded proteins from the cytoplasm is via fusion to a Tat-specific leader peptide.
  • the protein flux through the Tat export system is significantly lower than that of the more widely used Sec pathway. Consequently, the accumulation and steady state yield of proteins exported via the Tat pathway is low.
  • the prior art is thus deficient in methods of directing efficient export of folded proteins from the cytoplasm.
  • the present invention fulfills this long-standing need and desire in the art.
  • the present invention provides methods for the isolation of sequences that can serve as leader peptides to direct the export of heterologous proteins.
  • One aspect ofthe invention allows the isolation of leader peptides capable of directing proteins to the Tat secretion pathway. Further, the present invention discloses methods for identifying leader peptide mutants that can confer improved protein export.
  • the invention thus provides methods of identifying leader peptides that direct enhanced protein secretion in bacteria.
  • the methods disclosed herein comprise screening libraries of mutated leader peptides for sequences that allow rapid export and thus can rescue a short-lived reporter protein from degradation in the cytoplasm.
  • Leader peptides that mediate secretion through the Escherichia coli Twin Arginine Translocation (Tat) pathway, as well as those that direct other secretion pathways such as the sec pathway in bacteria can be isolated by the methods disclosed herein.
  • Mutant leader peptide sequences conferring improved export are also disclosed. The mutant leader peptides are shown to confer significantly higher steady state levels of export not only for the short lived reporter protein but also for other stable, long lived proteins.
  • a method of identifying leader peptides that direct increased protein export through pathways that include, but are not limited to, the Twin Arginine Translocation (TAT) pathway and the sec pathway.
  • TAT Twin Arginine Translocation
  • Such a method may involve constructing expression cassettes that place mutated leader peptides upstream of a gene encoding a short-lived reporter protein.
  • the short-lived reporter protein can be created by attaching a cytoplasmic degradation sequence to the gene encoding the reporter protein.
  • the resulting expression cassettes may then be expressed in bacteria, and reporter protein expressions in these bacteria measured. Mutated leader peptides expressed in cells that exhibit increased reporter protein expression comprise leader peptides that would direct increased protein export in bacteria.
  • a method of screening for a compound that inhibits or enhances protein export through pathways that include, but are not limited to, the Tat pathway and the sec pathway.
  • This method may comprise first constructing expression cassettes that place mutated leader peptides identified in the methods disclosed herein upstream of a gene encoding a short-lived reporter protein.
  • the short-lived reporter protein can be created by attaching a cytoplasmic degradation sequence to the gene encoding the reporter protein.
  • the resulting expression cassettes may then be expressed in bacteria, and reporter protein expression in these bacteria are measured in the presence or absence of the candidate compound. Increased reporter protein expression measured in the presence of the candidate compound indicates that the candidate compound enhances protein export, whereas decreased reporter protein expression measured in the presence of the candidate compound indicates that the candidate compound inhibits protein export.
  • a method for producing soluble and biologically-active heterologous polypeptide containing multiple disulfide bonds in a bacterial cell may involve constructing an expression cassette that places a leader peptide that directs protein export through the Twin Arginine Translocation pathway upstream of a gene encoding the heterologous polypeptide.
  • the heterologous polypeptide is then expressed in bacteria that have an oxidizing cytoplasm.
  • FIG. 1 shows the expression of green fluorescent protein in different plasmid constructs.
  • FIG. 1A shows minimal green fluorescent protein fluorescence in cells expressing pGFPSsrA, indicating that cytoplasmic SsrA-tagged green fluorescent protein is degraded almost completely.
  • FIG. IB shows enhanced green fluorescent protein fluorescence in cells expressing pTorAGFPSsrA, indicating improved green fluorescent protein export directed by the TorA leader peptide.
  • FIG. IC shows green fluorescent protein fluorescence in cells expressing pTorAGFP. The green fluorescent protein was expressed in both the cytoplasm and the periplasm.
  • FIG. 2 shows green fluorescent protein fluorescence in 6 different clones that exhibit increased Tat-dependent export due to mutated TorA leader peptides.
  • FIG. 3 shows periplasmic green fluorescent protein accumulation in the B6 and E2 clones.
  • FIG. 3 A shows western blot of green fluorescent protein in the periplasm (lanes 1- 3) and cytoplasm (lanes 4-6) of cells expressing the wild type construct (lanes 1 and 4), the B6 clone (lanes 2 and 5) and the E2 clone (lanes 3 and 6). GroEL is a cytoplasmic marker whereas DsbA is a periplasmic marker.
  • FIG. 3B shows periplasmic and cytoplasmic distribution of green fluorescent protein in cells expressing the wild type construct, the B6 clone and the E2 clone.
  • FIG. 4 shows increased green fluorescent protein fluorescence in cells expressing the wild type construct, the B6, B7 or E2 construct fused to untagged, proteolytically stable green fluorescent protein.
  • FIG. 5 shows western blot of green fluorescent protein in the periplasm (lanes 1-2), cytoplasm (lanes 3-4) and whole cell lysate (lanes 5-6) of cells expressing the wild type construct (lanes 1,3 and 5) or the B7 clone (lanes 2, 4 and 6).
  • GroEL is a cytoplasmic marker whereas DsbA is a periplasmic marker.
  • FIG. 6 Shows schematic of export of disulfide linked heterodimer in which only one polypeptide chain was fused to leader peptide.
  • FIG. 7 Shows western blot analysis and AP activity measurements for both periplasmic and cytoplasmic fractions in six genetic backgrounds.
  • the present invention provides methods of identifying and using leader peptides that direct enhanced protein secretion in bacteria.
  • Numerous proteins of commercial interest are produced in secreted form in bacteria. However, many proteins, including many antibody fragments and several enzymes of eucaryotic origin, cannot be exported efficiently through the main secretory pathway, the sec pathway, of bacteria.
  • An alternative pathway for the translocation of proteins from the cytoplasm of bacteria is called the "TAT" (twin-arginine-translocation) pathway. Whether a protein is directed to the sec machinery or the TAT pathway depends solely on the nature of the leader peptide, an amino acid extension of generally 15-30 residues located at the beginning of the polypeptide chain.
  • the leader peptide consists of three distinct regions: (1) the amino terminal n-region, (2) the hydrophobic core or h-region, and (3) the c- terminal region.
  • TAT-specific leader peptides A hallmark of both plant and prokaryotic TAT-specific leader peptides is the presence of the distinctive and conserved (S/T)-R-R-x-F-L-K (SEQ ID NO:l) sequence motif. This sequence motif is located at the n-region/h-boundary within leader peptides of known and predicted TAT substrates (Berks, 1996). Mutation of either arginine residue within the signal peptide significantly reduces the efficiency of protein translocation (Cristobal et al, 1999).
  • TAT-specific leader peptides are on average 14 amino acids longer due to an extended n-region and more basic residues in the c-region (Cristobal et al, 1999). However, the hydrophobic h-region in the TAT-specific leader peptides is significantly shorter due to a higher occurrence of glycine and threonine residues.
  • the twin-arginine (RR) motifs of wheat pre-23K and pre-Hcfl36 are essential for targeting by the thylakoid TAT pathway; this motif is probably a central feature of TAT signals.
  • the twin-arginine motif is not the only important determinant in TAT-specific targeting signals, and a further hydrophobic residue two or three residues after this motif seems also to be highly important.
  • Bacterial twin-arginine-signal peptides are similar to thylakoid TAT signals and can direct TAT-dependent targeting into plant thylakoids with high efficiency.
  • the vast majority of bacterial signal peptides contain conserved sequence elements in addition to the twin-arginine motif that imply special functions.
  • None of the known thylakoid twin-arginine signals contains phenylalanine at this position and only one (Arabidopsis P29) contains lysine as the fourth residue after the twin-arginine motif.
  • the bacterial cytoplasm contains a full complement of folding accessory factors, which can assist a nascent polypeptide in reaching its native conformation.
  • the secretory compartment of bacteria contains very few folding accessory factors such as chaperones and foldases. Therefore, for the production of many proteins, it is preferable for folding to occur first within the cytoplasm followed by export into the periplasmic space through the TAT system.
  • the acquisition of cofactors has to occur within the cytoplasm concomitant with folding. Consequently, cofactor- containing proteins must be secreted through the TAT pathway.
  • a limitation in the use of the protein secretion, and specifically of the TAT export pathway, for commercial protein production has been that the amount of protein that can be exported in this manner is low. In other words, the overall protein flux through the TAT system is substantially lower than that ofthe sec pathway.
  • the present invention thus provides, in one aspect, a method of identifying a leader peptide that directs increased protein export through the Twin Arginine Translocation or TAT pathway by constructing expression cassettes that put mutated candidate TAT- specific leader peptides upstream of a gene encoding a short-lived reporter protein.
  • a short lived reporter protein exhibits a decreased half life in the cytoplasm relative to reporter protein molecules that have been exported from the cytoplasm.
  • the short-lived reporter protein can be created, for example, by attaching a cytoplasmic degradation sequence to the gene encoding the reporter protein.
  • mutated leader peptides may be generated by random mutagenesis, error-prone PCR and/or site-directed mutagenesis.
  • the resulting expression cassettes can then be expressed in bacteria, and expression of the reporter protein be measured.
  • Mutated TAT-specific leader peptides expressed in cells that exhibit increased expression of reporter protein are leader peptides that would direct increased protein export through the TAT pathway.
  • Vectors ofthe invention include, but are not limited to, plasmid vectors and viral vectors.
  • green fluorescent protein may be used as a reporter protein.
  • the method takes advantage of the fact that functional, fluorescent green fluorescent protein can only be secreted using a TAT- specific leader peptide.
  • the export of green fluorescent protein via a TAT specific leader peptide is inefficient and results in the accumulation of an appreciable amount of precursor protein (green fluorescent protein with the TAT-specific leader) in the cytoplasm.
  • the cytoplasmic green fluorescent protein precursor protein is folded correctly and is fluorescent.
  • the cells exhibit high fluorescence, which in part is contributed by the cytoplasmic precursor and in part by the secreted, mature green fluorescent protein in the periplasm.
  • the overall high fluorescence of these cells contributes to a high background signal which complicates the isolation of leader peptide mutations that give rise to a higher flux of exported green fluorescent protein.
  • a short-lived version of green fluorescent reporter protein may be used. This short-lived version is rapidly degraded within the bacterial cytoplasm. Fusion of the SsrA sequence AANDENYALAA (SEQ ID NO: 119), for example, to the C-terminal of green fluorescent protein targets the protein for degradation by the ClpXAP protease system (Karzai et al, 2000). As a result, the half-life of green fluorescent protein in the cytoplasm is reduced from several hours to less than 10 min, resulting in a significant decrease in whole cell fluorescence.
  • mutant leader peptides were then screened for their ability to mediate enhanced protein secretion and rescue the short-lived green fluorescent protein from degradation in the cytoplasm, thereby leading to increased fluorescence ofthe bacteria. Clones exhibiting higher fluorescence were then isolated by flow cytometry.
  • the genetic screen described herein results in periplasmic-only accumulation of active reporter protein.
  • the mutated leader peptides direct folded green fluorescent reporter protein to the periplasm where the fluorescent protein remains active.
  • the SsrA C-terminal degradation peptide virtually all cytoplasmic green fluorescent protein is degraded.
  • the resulting cells glow green in a halo-type fashion due to the presence of periplasmic-only green fluorescent protein.
  • TAT-dependent export of green fluorescent protein that lacks the SsrA sequence would lead to green fluorescent protein accumulation in both the cytoplasm and the periplasm, resulting in substantial background signal that makes cell- based screening of GFP fluorescence impossible.
  • reporter proteins can be used in the methods of the present invention.
  • a person having ordinary skill in this art could readily isolate mutant leader peptides that result in higher levels of reporter protein expression in the periplasm in a number of ways. In one example, if the reporter is an
  • mutant leader peptides can be isolated by selecting on increasing concentrations of antibiotic.
  • the reporter is an immunity protein to a toxin (e.g., colicins)
  • mutant leader peptides can be isolated by selecting for resistance to toxin.
  • the reporter protein is a transport protein such as maltose binding protein
  • export ofthe transport protein is used to complement chromosomal mutants.
  • the chromogenic or fluoregenic substrate of a reporter enzyme e.g. alkaline phosphatase
  • the recombinant proteins are thus secreted into the periplasmic space or the growth medium in a functional and soluble form, alleviating problems associated with inclusion bodies and simplifying recovery. Furthermore, since proteins are folded and accumulate in the cytoplasm prior to TAT-dependent export, this export system will likely result in higher levels of active product accumulation within the host cell, thus maximizing the efficiency of the recombinant expression system.
  • the present invention can be applied in the development of technologies that capitalize on TAT-dependent export for combinatorial library screening and protein engineering applications.
  • improved cytoplasmic folding of disulfide bond containing proteins e.g., antibodies, eucaryotic enzymes
  • improved cytoplasmic folding of disulfide bond containing proteins can be assayed by fusion to optimized leader peptides that export the folded proteins of interest to the periplasm where it can be easily accessed by FACS-based or phage-based screening protocols.
  • the amount of active protein detected in the periplasm would be a quantitative indicator of the efficiency of folding in the cytoplasm.
  • the present invention of identifying and using leader peptides that direct enhanced protein secretion in bacteria is not limited to the TAT pathway.
  • the methods disclosed herein are equally applicable for identifying leader peptides that direct enhanced protein secretion through other secretion pathways as described above.
  • Signal sequences which promote protein translocation to the periplasmic space of Gram-negative bacterial are well- known to one of skill in the art.
  • the E. coli OmpA, Lpp, LamB, Mal ⁇ , PelB, and St ⁇ leader peptide sequences have been successfully used in many applications as signal sequences to promote protein secretion in bacterial cells such as those used herein, and are all contemplated to be useful in the practice of the methods of the present invention.
  • a person having ordinary skill in this art can readily employ procedures well- known in the art to construct libraries of mutated leader sequences and expression cassettes that inco ⁇ orate these mutated leader peptides, and screen these leader peptides according the methods described herein.
  • the present invention also relates to secretion of partially or fully folded cytoplasmic proteins with disulfide bonds.
  • the formation of disulfide bonds is essential for the correct folding and stability of numerous eukaryotic proteins of importance to the pharmaceutical and bioprocessing industries. Correct folding depends on the formation of cysteine-cysteine linkages and subsequent stabilization of the protein into an enzymatically active structure.
  • numerous studies have demonstrated that multiple disulfide bond-containing proteins cannot be expressed in active form in bacteria. Disulfide bond formation is blocked in the reducing environment of the cytoplasm of a cell due to the presence of thioredoxin reductase or reduced glutathione.
  • tissue plasminogen activator tPA
  • tPA tissue plasminogen activator
  • disulfide bonds form from specific orientations to promote correct folding of the native protein. Multiple disulfide bonds resulting from improper orientation of nascently formed proteins in the cell lead to misfolding and loss or absence of biological activity.
  • biologically- active polypeptide-containing multiple disulfide bonds produced according to the instant invention will be correctly folded; disulfide bonds will form to provide a tertiary and where applicable, quarternary structure leading to a molecule with native functional activity with respect to substrates and/or catalytic properties.
  • the proteins produced by the method disclosed herein are coreectly folded and biologically active without the need for reactivation or subsequent processing once isolated from a host cell.
  • proteins with multiple disulfide bonds can now be exported to the periplasm in a fully folded and therefore active conformation.
  • Complex proteins containing multiple disulfide bonds can be folded in the cytoplasm with the assistance of a full complement of folding accessory factors that facilitate nascent polypeptides in reaching their native conformation.
  • the folded proteins are then secreted into the periplasmic space or the growth medium in a functional and soluble form, thus alleviating problems associated with inclusion bodies and simplifying recovery.
  • active recombinant proteins accumulate simultaneously in two bacterial compartments (cytoplasm and periplasm), leading to greater overall yields of numerous complex proteins which previously could not actively accumulate in both compartments concurrently.
  • the present invention provides a method of producing at least one biologically-active heterologous polypeptide in a cell.
  • a leader peptide that directs protein export through the Twin Arginine Translocation pathway may be placed upstream of a gene encoding the heterologous polypeptide in an expression cassette.
  • the expression cassette can be expressed in a cell, wherein the heterologous polypeptide is produced in a biologically-active form.
  • the heterologous polypeptide is secreted from the bacterial cell, is isolatable from the periplasm or the culture supernatant of the bacterial cell, or is an integral membrane protein.
  • the heterologous polypeptide produced by this method can be a mammalian polypeptide such as tissue plasminogen activator, pancreatic trypsin inhibitor, an antibody, an antibody fragment or a toxin immunity protein.
  • the heterologous polypeptide may be a polypeptide in native conformation, a mutated polypeptide or a truncated polypeptide.
  • the above method can produce a heterologous polypeptide containing from about 2 to about 17 disulfide bonds.
  • This method may also produce two heterologous polypeptides that are linked by at least one disulfide bond.
  • the leader peptide comprises a sequence of SEQ ID NOs:25-46, 120-128 or a peptide homologous to SEQ ID NOs:25-46, 120-128.
  • Representative cells which are useful in this method include E. coli trxB mutants, E. coli gor mutants, or E. coli trxB gor double mutants such as E. coli strain FA113 or E. coli strain DR473.
  • the present invention also provides a series of putative TAT-specific leader peptides, which can be identified by a bioinformatics search from E. coli, cloned and examined for functional activities.
  • the present invention encompasses isolated leader peptides that direct protein secretion and export through the Twin Arginine Translocation pathway.
  • Representative leader peptides comprise sequences of S ⁇ Q ID NOs:25-46, 120- 128.
  • the present invention includes isolated TAT leader peptides that are homologous to S ⁇ Q ID NOs:25-46, 120-128.
  • the present invention also provides a method of identifying a leader peptide that directs increased protein export by constructing expression cassettes that put mutated leader peptides upstream of a gene encoding a short-lived reporter protein.
  • the short-lived reporter protein can be created by attaching a cytoplasmic degradation sequence to the gene encoding the reporter protein.
  • Representative cytoplasmic degradation sequences include S ⁇ Q ID NO:l 19, P ⁇ ST, or sequences recognized by LON, clPAP, clPXP, Stsh and HslUV.
  • the cytoplasmic degradation sequences are attached to either the N- or C-terminal of the reporter protein.
  • reporter proteins that can be used include fluorescent proteins, an enzyme, a transport protein, an antibiotic resistance enzyme, a toxin immunity protein, a bacteriophage receptor protein and an antibody.
  • Mutated leader peptides can be generated, for example, by random mutagenesis, error-prone PCR or site-directed mutagenesis, as well as other methods known to those of skill in the art.
  • the resulting expression cassettes can then be expressed in bacteria, and expression of the reporter protein measured.
  • Mutated leader peptides expressed in cells that exhibit increased expression of reporter protein comprise leader peptides that would direct increased protein export in bacteria.
  • This screening method is capable of identifying leader peptides that direct protein secretion through the general secretory (Sec) pathway, the signal recognition particle (SRP)-dependent pathway, the YidC-dependent pathway or the twin-arginine translocation (Tat) pathway.
  • the present invention also provides a method of screening for a compound that inhibits or enhances protein export in bacteria.
  • a leader peptide that directs protein export in bacteria may be placed upstream of a gene encoding a short-lived reporter protein in an expression cassette.
  • the expression cassette may then be expressed in bacteria in the presence or absence of a test compound. Increased expression of the reporter protein measured in the presence of the test compound indicates the compound enhances protein export, whereas decreased expression of the reporter protein measured in the presence of the compound indicates the compound inhibits protein export. Construction and examples of short-lived reporter protein are described above.
  • the present invention also provides a method of identifying a leader peptide that directs increased protein export through the Twin Arginine Translocation pathway by constructing expression cassettes that put mutated leader peptides specific for the Twin Arginine Translocation pathway upstream of a coding sequence encoding a short-lived reporter protein. Construction and examples of short-lived reporter protein are described above.
  • the mutated leader peptides can be generated by random mutagenesis, error-prone PCR or site-directed mutagenesis.
  • the resulting expression cassettes can then be expressed in bacteria, and expression of the reporter protein measured.
  • Mutated leader peptides expressed in cells that exhibit increased expression of reporter protein comprise leader peptides that would direct increased protein export through the Twin Arginine Translocation pathway. Examples of mutated leader peptides comprise sequences of SEQ ID Nos: 120-128.
  • a method of increasing export of heterologous polypeptide through the Twin Arginine Translocation pathway may be constructed that put mutated leader peptides identified according to the methods disclosed herein upstream of a gene encoding a heterologous polypeptide of interest. These expression cassettes may then be expressed in bacteria. Examples of mutated leader peptides comprise sequences of SEQ ID NOs:120-128.
  • the present invention also provides a method of screening for a compound that inhibits or enhances protein export through the Twin Arginine Translocation pathway.
  • a leader peptide specific for the Twin Arginine Translocation pathway may be placed upstream of a gene encoding a short-lived reporter protein in an expression cassette.
  • the expression cassette may then be expressed in bacteria in the presence or absence of a test compound. Increased expression of the reporter protein measured in the presence of the test compound indicates the compound enhances protein export, whereas decreased expression ofthe reporter protein measured in the presence of the compound indicates the compound inhibits protein export through the Twin Arginine Translocation pathway. Construction and examples of short-lived reporter protein are described above.
  • polypeptide or “polypeptide of interest” refers generally to peptides and proteins having more than about ten amino acids.
  • the polypeptides are "heterologous,” meaning that they are foreign to the host cell being utilized, such as a human protein produced by a CHO cell, or a yeast polypeptide produced by a mammalian cell, or a human polypeptide produced from a human cell line that is not the native source of the polypeptide.
  • Examples of a polypeptide of interest include, but are not limited to, molecules such as renin, a growth hormone (including human growth hormone), bovine growth hormone, growth hormone releasing factor, parathyroid hormone, thyroid
  • clotting factors such as factor NHIC, factor IX, tissue factor, and von Willebrands factor
  • anti-clotting factors such as Protein C, atrial naturietic factor, lung surfactant
  • a plasminogen activator such as human tPA or urokinase
  • mammalian trypsin inhibitor brain-derived neurotrophic growth factor, kallikreins, CT ⁇ F, gpl20, anti-HER-2, human chorionic gonadotropin, mammalian pancreatic trypsin inhibitor, antibodies, antibody fragments, protease inhibitors, therapeutic enzymes, lymphokines, cytokines, growth factors, neurotrophic factors, insulin chains or pro-insulin, immunotoxins,
  • bombesin thrombin, tumor necrosis factor- or ⁇ ,.enkephalinase, a serum albumin (such as
  • lactamase lactamase
  • Dnase Dnase
  • inhibin activin
  • NEGF vascular endothelial growth factor
  • receptors for hormones or growth factors integrin, protein A or D
  • rheumatoid factors neurotrophic factor
  • neurotrophin-3 neurotrophin-3, -4, -5, or -6
  • nerve growth factor such as ⁇ GF- ⁇
  • cardiotrophins cardiac hypertrophy factor
  • platelet-derived neurotrophin-1 platelet-derived neurotrophin-1
  • PDGF PDGF growth factor
  • fibroblast growth factor such as ⁇ FGF and ⁇ FGF
  • epidermal growth factor PDGF
  • fibroblast growth factor such as ⁇ FGF and ⁇ FGF
  • EGF growth factor
  • TGF transforming growth factor
  • TGF- ⁇ 3, TGF- ⁇ 4, or TGF- ⁇ 5 insulin-like growth factor-I and -H
  • des(l-3)-IGF-I brain IGF-I
  • insulin-like growth factor binding proteins CD proteins (such as CD-3, CD- 4, CD-8, and CD-I 9), erythropoietin, osteoinductive factors, bone morphogenetic proteins
  • BMPs interferons (such as interferon- ⁇ , - ⁇ , and - ⁇ ), colony stimulating factors (CSFs) (e.g., M-CSF, GM-CSF, and G-CSF), interleukins (Ils) (such as IL-1 to IL-10), superoxide dismutase, T-cell receptors, surface membrane proteins, decay accelerating factor, viral antigens such as a portion of the ADDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, antigens such as gpl20(IIIb), or derivatives or active fragments of any of the peptides listed above.
  • the polypeptides may be native or mutated polypeptides, and preferred sources for such mammalian polypeptides include human, bovine, equine, porcine, lupine, and rodent sources, with human proteins being particularly preferred.
  • TAT leader peptides were found using the Protein-Protein "BLAST" search engine available through the National Center for Biotechnology Information website. The following search strings were entered: SRRRFLK (SEQ ID NO:2),
  • SRRXFLX (SEQ ID NO:3), TRRXFLX (SEQ ID NO:4), SRRXXLK (SEQ ID NO:5),
  • SRRXXLA SEQ ID NO:6
  • TRRXXLK SEQ ID NO:7
  • TRRXXLA SEQ ID NO:8
  • SRRXXLT (SEQ ID NO:9), SRRXXIK (SEQ ID NO:10), SRRXXIA (SEQ ID NO:ll), SRRXFIX (SEQ ID NO:12), SRRXFMK (SEQ ID NO:13), SRRXFVK (SEQ ID NO:14),
  • SRRXFVA SEQ ID NO:15
  • SRRQFLK SEQ ID NO:16
  • RRXFLA SEQ ID NO:17
  • RRXFLK SEQ ID NO: 18
  • Searches were done for short, nearly exact matches and then screened for only those matches occurring within the first 50 residues of the protein while still maintaining the twin-arginines.
  • the first 100 residues of each leader peptide were then examined by "SignalP", a program for detecting Sec pathway leader peptides and cleavage sites (Nielsp e ⁇ f at, 1997).
  • the final list of putative TAT leader peptides is shown in Table 1. These peptides were cloned and examined for their abilities to direct secretion of a reporter protein, GFP-SsrA, through the TAT pathway.
  • Chloramphenicol (Cm) was used at the concentration of 50
  • strain XLI-Blue (recAl endAl gyrA96 thi-1 hsdRl 7 supE44 relAl lac
  • MC4100-P (MC4100 pcnBl) and B1LK0-P (MC4100 AtatCpcnBl).
  • leader peptide DNA sequence was first subcloned into pKKGS (DeLis ' ' ⁇ !, " 2 ⁇ 5), which is based on the low-copy pBAD33 plasmid (Guzman et al, 1995). Standard methods were used to amplify DNA and Qiagen kits were used for all DNA purification steps.
  • Each leader peptide gene was first PCR amplified from XLI-Blue genomic DNA using a forward primer that contained a S ⁇ cl cleavage site and a reverse primer that contained an Xbal cleavage site. Forward primers were designed to incorporate at least the first 18 nucleotides of the leader peptide.
  • Cells were pelleted by centrifugation at 5000 x g, resuspended in 1 ml of cell fractionation buffer (30 mM Tris-HCl, pH 8.0, 20% (w/v) sucrose, 1 mM Na 2 EDTA), and incubated at 25°C for 10 min. The cells were again centrifuged at 5000 x g, the supernatant discarded, and the pellet resuspended in 133 ⁇ l of ice-cold 5 mM MgSO . After 10 min on ice, the cells were centrifuged at full speed, and the supernatant was retained as the periplasmic fraction. The pellet was resuspended in 250 ⁇ l of PBS and sonicated for 30 seconds. The cells were centrifuged at full speed and the supernatant was retained as the cytoplasmic fraction.
  • cell fractionation buffer 30 mM Tris-HCl, pH 8.0, 20% (w/v) sucrose, 1 mM Na
  • leader peptide-GFP-SsrA constructs To express the leader peptide-GFP-SsrA constructs, overnight (o/n) cultures of MC4100-P and B1LK0-P containing each of the 30 plasmids were grown in LB media as described above. Single colonies were grown overnight in 2 mL of media. Five hundred
  • TAT leader peptides were screened in a genetic screen as described previously (DeLisa et al 2002). With this genetic screen, a leader peptide that directs GFP through the TAT pathway would be fluorescent in tatC + cells (MC4100-P) but non- fluorescent in tatC cells (B1LK0-P) since tatC is absolutely necessary for TAT export. By contrast, a leader peptide that directed GFP to the periplasm via the Sec pathway would be non-fluorescent in both types of cells. Of note is the use of E.
  • coli strains containing a mutation ia pcnBl which lowers the copy number of those plasmids (such as pBAD18- Cm) that contain the pBR322 replicon.
  • pBAD18-Cm which is normally a high copy vector, is only present at approximately 5-10 copies per cell in pcnBl mutants. This system proved optimal for use with the TAT pathway genetic screen.
  • the FACS analysis for the pBAD18-Cm constructs are shown in Table 4 (a list of arithmetic mean fluorescence values).
  • the FACS data for the pBAD18-Cm constructs shows that six leader peptides (BisZ, , NapA, NapG, Yael, YgfA, and YggJ) gave inconclusive GFP export through the TAT pathway (low signal in both wt and tatC mutant celsl) while at least 17 (AmiC, DmsA, FdnG, FdoG, FhuD, HyaA, HybA, NrfC, Sufi, TorA, WcaM, YacK, YahJ, YdcG, YdhX, YfhG, and, YnfE) are capable of exporting GFP via the TAT pathway.
  • NrfC NrfC MTWSRRQFLTGVGVLAAVSGTAGRVVAK 26 YahJ MKESNSRREFLSQSGKMNTAAALFGTSVPLAHAA 27
  • WcaM rev2 59.7 GCGATGTCTAGAGCTTTGTCGGGCGGG 83 AAG
  • YfhG rev2 53.4 GCGATGTCTAGACGTATCAATGGCTGG 91 CTT
  • YcdB rev2 52.1 GCGATGTCTAGACGCACTTTGCGTTTTT 92 TG
  • FhuD rev4 53.3 GCGATGTCTAGAATTGGGATCAATAGC 95 CGC
  • Ygf rev2 50.6 GCGATGTCTAGAGAATACAGCGACCGT 96 ATG
  • BisZ rev2 52.3 GCGATGTCTAGATTTACCGCCCTTCTCT 97 TC
  • E. coli strain XLI-Blue (recAl endAl gyrA96 thi-1 hsdRl 7 supE44 relAl lac [F' proAB
  • lacPZ ⁇ M15TnlO (Tet 1 )] was used for all experiments unless otherwise noted.
  • E. coli XLl -Blue tatB and XLI-Blue tatC were made using pFAT24 (Sargent et al. 1999) and pFAT166 (Bogsch et al, 1998) respectively according to established procedure (Bogsch et al, 1998). Strains were routinely grown aerobically at 37°C on Luria-Bertani (LB)
  • media and antibiotic supplements were at the following concentrations: ampicillin, 100 ⁇ g
  • Plasmid pGFP was constructed by cloning the GFPmut2 variant (Crameri et al, 1996) using the primers GFPXbal (5'-GCGATGTCTAGAAGTAAAGG AGAAGAACTTTTCACT-3', SEQ ID NOT 12) and GFPHindm (5'- GCGATGAAGCTTCTATTTGTATAGTTCATCCAT-3', SEQ ID NO:113) which introduced unique restriction sites of .
  • Plasmid pGFPSsrA was made similarly using the primers GFPXbal and GFPSsrA (5 '-GCGATGAAGCTTGCATGCTTAAGCTGCTAAAGCGTAGTTTTCG
  • Plasmid pTorAGFP and pTorAGFPSsrA were made by PCR amplification of E. coli genomic DNA using primers TorASacI (5'- GCGATGGAATTCGAGCTCTTAAAGAGGAGAAAGGTCATGAACAATAACGATCT CTTTCAG-3', SEQ ID NOT 15) and TorAXbal (5'-
  • a library of random mutants was constructed by enor prone PCR of the torA gene sequence using 3.32 or 4.82 mM Mg 2+ (Fromant et al, 1995), XLI-Blue genomic DNA and the following primers: torASacI (5'- GCGATGGAATTCGAGCTCTTAAAGAGGAGAAAGGTCATGAACAATAACGATCT CTTTCAG-3') (SEQ ID NOT 17) and torAXbal (5'-
  • EXAMPLE 5 Library Screening Transformants were grown at 37°C in LB medium with chloramphenicol, induced with 0.2% arabinose for 6 h and diluted 200-fold in 1 ml PBS. FACS gates were set based upon FSC/SSC and FL1/FL2. Prior to sorting, the library cell population was labeled with propidium iodide for preferential labeling of non- viable cells. A total of ca. 3xl0 6 cells were analyzed by flow cytometry and 350 viable cells were collected. The collected solution was filtered, and the filters were placed on LB plates with chloramphenicol. After a 12 hour incubation at 37°C, individual colonies were inoculated into LB with chloramphenicol in triplicate 96-well plates.
  • the fraction of periplasmic proteins was obtained by spheroplasting bacteria by lysozyme-EDTA treatment under isotonic conditions according to the procedure of Kaback (1971). Briefly, cells were collected by centrifugation and resuspended to an OD 600 of
  • Lysozyme (Sigma) was added to 50 ⁇ g/ml, and cells were incubated for 1 h at room temperature to generate spheroplasts.
  • the spheroplasts were pelleted by 15 min of centrifugation at 3,000 x g, and the supernatant containing periplasmic proteins was collected for electrophoretic analysis.
  • the pellet containing spheroplasts was resuspended in 10 ml of TE (10 mM Tris-Cl [pH 7.5], 2.5 mMNa-EDTA) and homogenized in a French press cell (Carver) at 2,000 lb/in .
  • TE Tris-Cl [pH 7.5], 2.5 mMNa-EDTA
  • the plasmid pTorAGFP contains a gene encoding the TAT-specific leader peptide and the first eight amino acids of the E. coli trimethylamine N-oxide reductase (TorA) fused to the FACS optimized GFPmut2 gene (Crameri et al, 1996).
  • the TorA-GFP gene was placed downstream of the arabinose-inducible promoter pBAD. Cells induced with arabinose for 6 hours and analyzed by FACS gave a mean fluorescence intensity (MFL1) above 500 arbitrary units (FIG. IC). In agreement with previous reports (Santini et al, 2001), cell fractionation by osmotic shock revealed that ca.
  • a nucleotide sequence encoding a C-terminal SsrA degradation peptide was fused to the TorA-GFP gene.
  • the resulting gene, pTorA-GFP-SsrA was also placed downstream from a pBAD promoter in the vector pTorAGFPSsrA.
  • GFP without the leader peptide was fused in frame to the SsrA tag and expressed from the plasmid pGFPSsrA.
  • GFP-SsrA-expressing cells showed virtually no appreciable fluorescence intensity, indicating that cytoplasmic SsrA-tagged GFP is degraded almost completely (FIG. 1 A).
  • Cells expressing TorA-GFP-SsrA were ca.
  • FIG. 3 Representative Western blots indicate that periplasmic GFP accumulation by cells expressing the B6 and E2 clones was significantly increased relative to those expressing wild type construct (lanes 1-3, FIG. 3). Furthermore, there was virtually no detectable GFP protein in the cytoplasmic fractions. This was because the presence of the SsrA tag resulted in degradation of the protein. Also shown in FIG. 3 were Western bands of two fractionation marker proteins, the cytoplasmic marker GroEL and the periplasmic marker DsbA. The absence of GroEL in the periplasmic fraction and the high level of DsbA in periplasmic fractions confirm that cell fractionation was successful.
  • FIG. 3B Data on the distribution of fluorescence in the cytoplasmic and periplasmic fractions for two mutant TorA leader peptides are shown in FIG. 3B. Nearly identical results were observed for the remaining four clones (B7, FI, FI 1 and H2). The sequences of the six clones were determined and indicated that in all cases either one or two single residue mutations were sufficient to alter the observed export dynamics. In general, these mutations occur within or in close proximity to the conserved S/T-R-R-x-F-L-K (SEQ ID NOT) consensus motif (Table 6).
  • TorA leader peptides confer increased GFP export not only when the protein is tagged with the SsrA tag but also for the untagged, proteolytically stable GFP (FIG. 4). This increase in fluorescence was due to the increased periplasmic flux of folded GFP protein. Similar results were observed for the remaining clones fused to GFP.
  • the intense band corresponding to mature GFP accumulated by the TorAB7-GfP construct signifies substantially more periplasmic processing of GFP relative to wild type TorAGFP cells (FIG. 5, lanes 5 and 6). Similar results were observed for all five remaining clones. As described above, the GroEL and DsbA marker proteins confirm successful cell fractionations.
  • Twin arginine consensus motif is indicated by underlined amino acids; first 8 residues of mature TorA protein are indicated by italics; mutations in TorA leader peptide are indicated by emboldened letters.
  • TAT-specific leader peptide TorA may be fused to alkaline phosphatase (TorA- PhoA fusion).
  • Alkaline phosphatase (PhoA) contains two disulfide linkages that are consecutive in the primary sequence so that they are normally incapable of forming in the cytoplasm of E. coli strains having reducing environment (e.g. strain DHB4). Since the TAT pathway requires folded or at least partially folded substrates, TAT-dependent secretion of PhoA in DHB4 cells would be blocked due to the accumulation of unfolded PhoA in the cytoplasm.
  • the bacterial cytoplasm is maintained in a reduced state due to the presence of reducing components such as glutathione and thioredoxins that strongly disfavors the formation of disulfide bonds within proteins.
  • reducing components such as glutathione and thioredoxins that strongly disfavors the formation of disulfide bonds within proteins.
  • coli depends on aerobic growth in the presence of either of the two major thiol reduction systems: the thioredoxin and the glutathione-glutaredoxin pathways. Both the thioredoxins and the glutaredoxins are maintained in a reduced state by the action of thioredoxin reductase (TrxB) and glutathione, respectively. Glutathione is synthesized by the gshA and gshB gene products. The enzyme glutathione oxidoreductase, the product of the gor gene, is required to reduce oxidized glutathione and complete the catalytic cycle ofthe glutathione-glutaredoxin system.
  • TrxB thioredoxin reductase
  • glutathione reductase glutathione reductase
  • trxB null mutant stable disulfide bonds can form in normally secreted proteins, such as alkaline phosphatase, when they were expressed in the cytoplasm without a signal sequence.
  • the two thioredoxins were oxidized in the trxB mutant and served as catalysts for the formation of disulfide bonds. Disulfide bond formation was found to be even more efficient in double mutants defective in both the thioredoxin (trxB) and glutathione (gor or gshA) pathways.
  • the resulting ahpC* allele allows efficient growth in normal (non-reducing media) without compromising the formation of disulfide bonds in the cytoplasm.
  • trxB, gor ahpC* mutant strains (such as E. coli DR473 or FA113) exhibit the ability to support disulfide bond formation in the cytoplasm and also can grow equally well as the corresponding wild-type strain DHB4 in both rich and minimal media.
  • TorA leader peptide was fused to a truncated version of tissue plasminogen activator (vtPA) consisting of the kringle 2 and protease domains with a total of nine disulfides (TorA-vtPA), or to a heterodimeric 2610 anti- digoxin antibody fragment with 5 disulfide bonds including an interchain disulfide linkage (TorA-Fab).
  • vtPA tissue plasminogen activator
  • DR473 cells expressing TorA-vtPA and TorA-Fab showed remarkably high levels of activities in cell lysates for each of the expressed proteins relative to DHB4 cells expressing identical constructs. Activities in DHB4 lysates were virtually undetectable in all cases except for vtPA. Fractionation experiments further confirmed that significant portion (30-50%) of the overall activities for each of the proteins was found in the periplasmic fraction.
  • PhoA consists of two polypeptide chains with a total of two disulfide bonds that are required for folding and enzymatic activity while Fab is comprised of two non-identical chains (each with two intermolecular disulfide bonds) linked together by an intermolecular disulfide bond. Normally, the formation of disulfide bonds in these proteins occurs following export into the oxidizing environment of the periplasmic space.
  • strains and plasmids used are described in Table 7.
  • Strains DHBA and DRA were obtained by PI transduction of the dsbAv.kanl allele from JCB571 (MC1000 phoR zihl2::Tn!0 dsbAv.kan) into E. coli strains DHB4 and DR473, respectively.
  • Strain D0D was obtained by PI transduction of tatB::kan allele from JCB571 (MC1000 phoR zihl2::Tn!0 dsbAv.kan) into E. coli strains DHB4 and DR473, respectively.
  • Strain D0D was obtained by PI transduction of tatB::kan allele from
  • E. coli strain XLI-Blue (recAl endAl gyrA96 thi-1 hsdR17 supE44 relAl lac [F' proAB lacPZDM15 TnlO (Tet 1 )] was used for cloning and plasmid propagation.
  • phosphatase assays cells were subcultured from overnight cultures into minimal M9 medium [M9 salts with 0.2% glucose, 1 ⁇ g/ml vitamin Bl, 1 mM MgSO 4 , 50 ⁇ g/ml 18 amino acids (excluding methionine and cysteine)] at a 100-fold dilution, and then incubated at 37°C.
  • M9 salts with 0.2% glucose, 1 ⁇ g/ml vitamin Bl, 1 mM MgSO 4 , 50 ⁇ g/ml 18 amino acids (excluding methionine and cysteine)] at a 100-fold dilution, and then incubated at 37°C.
  • Fab studies cells were subcultured from overnight cultures into fresh LB medium (5% v/v) and then incubated at 30°C. Growth was to mid-log phase (OD 6 oo ⁇ 0.5) and induction of both alkaline phosphatase and Fab was accomplished by addition of IPTG to a final concentration of 0.1 m
  • Plasmid p33RR was constructed by PCR amplification of the E. coli torA signal sequence (ssTorA) from E. coli genomic DNA using primers TorASacI and TorAXbal described above. Amplified DNA was digested using S ⁇ cl and Xbal and inserted into the same sites of pBAD33.
  • Plasmid p33KK was generated identically as p33RR except that mutagenic primer TorAkk (5'- gcgatggagctcttaaagaggagaaaggtcatgaacaataacgatctctttcaggcatcaaagaaacgttttctggcaactc-3') (S ⁇ Q ID NO: 129) was used to PCR amplify the torA signal sequence.
  • DNA encoding signal sequence-less phoA (PhoA ⁇ 2-22) was generated by PCR amplification from E. coli
  • a DNA fragment encoding torA signal sequence (or torA (R11K;R12K) signal sequence) fused in- frame to phoA was amplified from plasmid p33RRP (or p33KKP) using primers TorASacI (or TorAKK) and Phorev.
  • the PCR amplified DNA was digested with BspRl and Hmdm and inserted into the Nc ⁇ l-Hz ' r ⁇ di ⁇ sites of pTrc99 resulting in plasmid pRRP (or pKKP). Constraction of alkaline phosphatase fusions to alternate signal sequences (e.g.
  • Plasmid pTorA-Fab was constructed by PCR amplification of the anti-digoxin dicistronic Fab gene encoded in pTrc99-Fab (Levy et al, 2001) using primers Fab for (5'-gctgctagcgaagttcaactgcaacag-3') (S ⁇ Q ID NO: 132) and Fabrev (5'-gcgatgcccgggggctttgttagcagccggatctca-3') (S ⁇ Q ID NO: 133) and amplification of torA signal sequence was with primers TorASacI and TorAover (5'-gcgctgttgcagttgaacttcgctagcagcgtcagtcgccttg-3') (S ⁇ Q DD NO.T34).
  • the two PCR products were fused via overlap extension PCR using primers TorASacI and Fabrev.
  • the overlapped product was digested with BspRl and Xma ⁇ and inserted into the Ncol andXmal sites of pTrc99A. All plasmids were confirmed by sequencing. Cell fractionations:
  • the fraction of periplasmic proteins was obtained by ice-cold osmotic shock (Sargent et al, 1998). Specifically, cells were collected by centrifugation and resuspended in buffer containing 30 mM Tris- ⁇ Cl (p ⁇ 8.0), 0.5 M sucrose, 1 mM ⁇ a- ⁇ DTA and 20 mM iodoacetamide was used to prevent spontaneous activation of alkaline phosphatase. Cells were incubated for 10 min at 25°C followed by centrifugation for 10 min at 5000xg and 4°C. Pellets were then resuspended in ice-cold 5 mM MgSO 4 and kept on ice for 10 min.
  • nitrophenyl phosphaste (pNPP; Sigma) solution (1 fast tablet in 100 mM Tris-HCl, pH 7.4)
  • Assays were performed as follows. Ninety-six-well high binding assay plates (Corning-Costar) were coated (100 ul well) with 4 ug ml "1 BSA-digoxin conjugate or with 4 ug ml "1 BSA (100 ul/well). Coated plates were blocked overnight at 4°C with 5% nonfat dry milk in PBS. The presence of anti-digoxin scFv and Fab antibodies was detected using rabbit-anti-mouse IgG (specific to (Fab') 2 light chains) diluted 1:2000 followed by goat anti-rabbit IgG (H + L) conjugated with horse radish peroxidase diluted 1:1000. Development was with addition of OPD substrate (Sigma) and the reaction was quenched by addition of 4.5 N H 2 SO . Plates were read at 490 nm on a Bio-Tek Instruments microplate reader.
  • coli cells harboring plasmid pTorA-AP and induced with IPTG (0.1 mM) produced large quantities of cytoplasmic AP as detected by Western blotting.
  • AP activity in the cytoplasmic fraction of DHB4 cells was almost entirely inactive due to its failure to acquire disulfides bonds in the cytoplasm of this strain.
  • a trxB gor ahpC triple mutant of E was critical for Tat-dependent export.
  • ⁇ -galactosidase (LacZ) activity in subcellular fractions was measured (see above) and only samples with ⁇ 5% LacZ activity in the periplasm were analyzed herein.
  • ⁇ -galactosidase (LacZ) activity in subcellular fractions was measured (see above) and only samples with ⁇ 5% LacZ activity in the periplasm were analyzed herein.
  • cross-reaction of the cytoplasmic chaperone GroEL with specific antisera was used as a control for subcellular fractionation.
  • the folding status of AP was the major determinant in the ability to export this protein by the Tat pathway.
  • ssTorA(RHK;R12K)-AP accumulated in the cytoplasm of DHB4 cells to a much lesser extent than ssTorA-AP in the same cells.
  • a proper Tat signal (Arg-Arg) targets even misfolded AP to the cytoplasmic side of the inner membrane.
  • membrance localization sequesters some of the misfolded enzyme from proteolysis.
  • the defective Lys-Lys leader peptide does not properly interact with the Tat machinery and as a result non-targeted AP is more susceptible to cytoplasmic proteolysis.
  • D0D (DR473 tatBv.kan).
  • AP was exported in a Tat-dependent fashion when fused to two different signal sequences from formate dehydrogenase-N (FDH-N) subunit G (ssFdnG) and FDH-O subunit G (ssFdoG). Cnller.tivelv, these results confirm that the appearance of AP in the periplasm was completely dependent on export via the Tat pathway and that translocation could be
  • the ssTorA-AP fusion protein was produced in an E. coli dsbA null mutant (strains DHBA and DRA).
  • DsbA is the major periplasmic enzyme involved in catalyzing disulfide bond formation in newly synthesized proteins normally secreted by the Sec pathway.
  • both the DHBA and DRA mutant strains were completely unable to oxidize periplasmic proteins due to a null mutation of dsbA.
  • Tat leaders AmiA, FdnG, FdoG, HyaA, H bA and TorA were unable to export AP when the cytoplasm was reducing; however 2) certain other Tat leader peptides (DmsA, Sufi, YacK and YcbK) could direct AP to the periplasm even though disulfide bond formation in the cytoplasm was not possible. This was likely due to Sec-dependent export of AP. As expected, nearly all of the leader peptides were able to direct AP to the periplasm of strain DR473 due in part to the more oxidizing cytoplasm.
  • PhoA is translocated as a monomer (-48 kDa) or in its active homodimeric state (-96 kDa) is still unclear, although PhoA is known to fold rapidly into its highly stable, native dimeric state.
  • the notion that the large alkaline phosphatase dimer is compatible with the Tat machinery is supported previous studies demonstrating that the 142 kDa FdnGH subcomplex of E. coli formate dehydrogenase-N is transported by the Tat system.
  • a considerable portion of the proteins exported by the Tat pathway are enzymes that acquire cofactors in the cytoplasm prior to export and generally function in respiratory or electron transport processes (e.g., E. coli trimethlamine N-oxide reductase).
  • E. coli trimethlamine N-oxide reductase The acquisition of cofactors in the cytoplasm requires tertiary structure contacts that occur only after folding has been largely completed.
  • membrane targeting and the acquisition of nickel by HybC the large subunit of the E. coli hydrogenase 2
  • HybO which contains a Tat-specific leader peptide.
  • the model favored is that the small and large subunits of hydrogenase 2 first form a complex in the cytoplasm and the complex is then targeted to the membrane by virtue of the leader peptide of the small subunit.
  • a non-physiological heterodimeric antibody fragment could be exported via the Tat translocator when folded properly in the cytoplasm.
  • the Tat pathway could also export a disulfide linked heterodimer in which only one polypeptide chain was fused to the TorA leader peptide (see schematic, FIG. 6).
  • a Fab antibody fragment specific for the cardiac glycoside digoxin was used which consisted of two polypeptide chains, the heavy and light chains, linked together via a disulfide bond. In addition, the heavy and light chains each contained two intramolecular disulfide bonds.
  • the TorA leader peptide was fused only to the heavy chain (V H -C H which was co-expressed with the light chain (Ni- ) from a dicistronic operon. In this fashion, the TorA-heavy chain carries the light chain into the periplasm in a 'piggyback' fashion only if the interchain disulfide bridge is formed first in the cytoplasm prior to translocation.
  • ⁇ ssDsbC resulted in a significant increase in the amount of Fab in the periplasm (-50% in the osmotic shock fraction). This may be due to co-expression of chaperones in the cytoplasm increasing the amount of protein competent for export presumably because it improved the yield of folded protein.
  • Fab was immunologically probed using a primary antibody that recognizes mouse light chain sequences. Therefore, the bands seen confirmed that the light chain was properly recruited by the heavy chain via intermolecular disulfide bond formation and subsequently delivered to the periplasmic space.
  • the localization of the cytoplasmic marker protein GroEL and the periplasmic marker protein DsbC demonstrates that the subcellular fractionation was successful.
  • the Fab protein in the periplasmic fraction of DRA cells was correctly folded and functional as evidenced by its ability to bind the antigen, digoxin in ELISA assays.
  • the Tat pathway is capable of exporting a fully oxidized Fab across the membrane and (ii) the process is dependent on the assembly ofthe light and heavy chains and the formation ofthe intermolecular disulfide within the cytoplasm prior to export.
  • the transport of oxidized, presumably fully folded, Fab molecules into the periplasm provides conclusive evidence for the hitchhiker mode of export suggested previously whereby a polypeptide containing a Tat leader peptide mediates the translocation of a second leaderless polypeptide with which it associates in the cytoplasm.

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Abstract

L'invention concerne des procédés d'isolement de peptides leader pouvant diriger l'exportation de protéines hétérologues à partir du cytoplasme bactérien. Lesdits procédés se fondent sur le criblage de bibliothèques de peptides leader putatifs ou de mutants de peptides leader pour des séquences qui permettent une exportation rapide et peuvent ainsi empêcher la dégradation d'une protéine reporter à durée de vie courte dans le cytoplasme. Lesdits peptides leader mutants identifiés dans la présente invention peuvent conférer de manière significative des niveaux d'états stables d'exportation plus élevés non seulement pour une protéine reporter à durée de vie courte mais aussi pour d'autres protéines stables à durée de vie longue. Lesdits peptides leader peuvent être utilisés pour diriger ou augmenter la sécrétion des protéines. L'invention concerne de plus des procédés d'exportation de protéines pliées de manière cytoplasmique par l'intermédiaire d'une voie Tat. Des protéines présentant des liaisons disulfure sont pliées en premier à l'intérieur du cytoplasme dans des souches mutantes appropriées oxydantes. Lesdites protéines pré-pliées de manière cytoplasmique qui contiennent des liaisons disulfure sont alors exportées par l'intermédiaire de la voie Tat.
EP02795597A 2001-11-05 2002-11-05 Genie de peptides leader pour la secretion de proteines recombinees dans des bacteries Withdrawn EP1451367A4 (fr)

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EP2548030B1 (fr) * 2010-03-18 2015-05-27 Cornell University Préparation d'anticorps correctement pliés utilisant la présentation sur la membrane intérieure d'intermédiaires de translocation twin-arginine
CN103797122A (zh) * 2011-04-08 2014-05-14 安瑟生物科技私人有限公司 用于大肠杆菌中异源蛋白质生产的新的表达和分泌载体系统
KR101470595B1 (ko) 2012-08-01 2014-12-10 대구가톨릭대학교산학협력단 세포외로 분비되는 녹색형광단백질, 이를 코딩하는 유전자 및 이 유전자 발현을 위한 벡터
CN102851270A (zh) * 2012-08-03 2013-01-02 江南大学 一种杂交链霉菌胰蛋白酶酶原及其应用
GB201713732D0 (en) * 2017-08-25 2017-10-11 Alta Innovations Ltd Tat expression system
CN109575116B (zh) * 2018-11-09 2022-04-22 广东海洋大学 一种线粒体定位导肽及其发现方法与应用
CN110616227A (zh) * 2019-09-30 2019-12-27 天津科技大学 一种来源于黄粉虫的抗冻蛋白的基因、重组表达载体、工程菌株和应用

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AU3243199A (en) * 1998-03-05 1999-09-20 University Of British Columbia, The Methods for assaying type iii secretion inhibitors
EP1068339B1 (fr) * 1998-04-01 2008-07-23 The Governors of the University of Alberta Compositions et methodes de secretion proteique
DK1356060T3 (da) * 2000-09-18 2006-05-01 Genencor Int Dobbeltarginintranslokation i Bacillus
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CN100564540C (zh) 2009-12-02
WO2003040335A2 (fr) 2003-05-15
WO2003040335A3 (fr) 2003-12-31
CN1788092A (zh) 2006-06-14

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