WO2002061041A2 - Solubility reporter gene constructs - Google Patents
Solubility reporter gene constructs Download PDFInfo
- Publication number
- WO2002061041A2 WO2002061041A2 PCT/US2001/051426 US0151426W WO02061041A2 WO 2002061041 A2 WO2002061041 A2 WO 2002061041A2 US 0151426 W US0151426 W US 0151426W WO 02061041 A2 WO02061041 A2 WO 02061041A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cell
- solubility
- protein
- host cell
- target polypeptide
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Definitions
- This invention pertains to the field of drug discovery and in particular, compositions and methods that aid the drug discovery process.
- heat shock proteins indicate that there exists a relationship between some of these proteins and protein folding. It is well known that cells which are subjected to elevated temperature respond by inducing the expression of a set of genes known as heat shock genes.
- the proteins encoded by these genes, the heat shock proteins provide functions that help to control the deleterious effects of the elevated temperature and include chaperones and protease molecules.
- the heat shock response has been studied in detail in both eukaryotic and prokaryotic systems and is highly conserved throughout evolution. A thorough analysis of the genes induced by heat shock has been performed on the genome of the Gram negative bacterium E. coli to identify a set of genes induced by this stimulus (Richmond et al. (1999) Nucleic Acids Res.
- the present invention provides cells, reagents, and methods for determining whether a host cell expresses a polypeptide of interest in soluble or insoluble form.
- the invention provides host cells that contain: a) a solubility reporter nucleic acid that includes a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide-expressing nucleic acid that includes a polynucleotide that encodes a target polypeptide. Expression of the target polypeptide in an insoluble form causes a change in expression of the reporter gene.
- the solubility responsive promoter is upregulated when the target polypeptide is expressed in insoluble form in some embodiments of the invention; in other embodiments the solubility responsive promoter is downregulated when the target polypeptide is expressed in insoluble form.
- Arrays of two or more populations of such host cells are also provided; the host cells of each population differ in the target polypeptides expressed by the host cells.
- the invention also provides methods for determining the solubility of a target polypeptide.
- These methods involve culturing host cells that contain: a) a solubility reporter nucleic acid that includes a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide-expressing nucleic acid that includes a polynucleotide that encodes a target polypeptide under conditions in which the target polypeptide is expressed.
- the solubility of the expressed target polypeptide is then determined by detecting whether expression of the reporter gene is increased or decreased.
- Additional embodiments of the invention provide methods for identifying mutations in a cell that alter the solubility of a target polypeptide. These methods involve: a) treating a cell with a mutagen; b) introducing into the cell a solubility reporter nucleic acid that includes a protein solubility responsive promoter operably linked to a reporter gene and a target polypeptide-expressing nucleic acid that includes a polynucleotide that encodes a target polypeptide; c) culturing the cell under conditions favorable for expression of the target polypeptide; d) measuring expression of the reporter gene; and e) comparing the level of expression of the reporter gene in the cell with the level observed in an unmutated cell that also contains the solubility reporter nucleic acid and the target polypeptide- expressing nucleic acid to identify a cell that comprises a mutation that alters the solubility of the target polypeptide.
- the invention provides methods for identifying alterations to a polynucleotide that encodes a target polypeptide that alter the solubility of the target polypeptide. These methods involve: a) altering a polynucleotide that encodes the target polypeptide to form an altered polynucleotide; b) introducing into a cell a solubility reporter nucleic acid that includes a protein solubility responsive promoter operably linked to a reporter gene, and a target polypeptide-expressing nucleic acid that includes the altered polynucleotide; c) culturing the cell under conditions favorable for expression of the target polypeptide; d) measuring the expression of the reporter gene; and e) comparing the level of expression of the reporter gene with the level observed in a cell with an unaltered polynucleotide that encodes the target polypeptide, to identify an alteration to the polynucleotide that changes the solubility of the encoded target polypeptide
- the invention also provides methods for identifying variations in a process for biosynthesis of a target polypeptide that alter the solubility of the target polypeptide. These methods involve culturing a host cell under alternative conditions in which the target polypeptide is expressed.
- the host cell includes: a) a solubility reporter nucleic acid that comprises a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide-expressing nucleic acid that includes a polynucleotide that encodes a target polypeptide. Expression of the reporter gene by host cells grown under each of the alternative conditions is then compared to determine which condition results in a desired level of solubility of the target polypeptide.
- the invention also provides methods for identifying an antibiotic agent.
- the methods involve: a) contacting a cell that contains a solubility reporter nucleic acid with a candidate antibiotic agent, wherein the solubility reporter nucleic acid includes a protein solubility responsive promoter operably linked to a reporter gene; and detecting the level of expression of the reporter gene.
- a change in the expression level of the reporter gene in a cell contacted with the candidate antibiotic agent, compared to reporter gene expression level in a cell which is not contacted with the candidate antibiotic agent, is indicative of an agent that inhibits protein folding in the cell.
- the present invention also provides polynucleotides that include a protein solubility responsive promoter which is operably linked to a polynucleotide that encodes a detectable or selectable product.
- the polynucleotide can further comprise an expression construct for a target protein.
- This invention also provides a solubility reporter system that includes these solubility reporter polynucleotides together with an expression construct for a target protein.
- the invention also provides gene delivery vehicles and expression vectors and host or genetically modified cells containing at least polynucleotides of the invention and the genetic reporter system.
- Figure 1 shows the promoters of known heat shock genes that were induced during the expression of insoluble protein.
- the nucleotide sequences were aligned manually, allowing one gap in the sequence. Sequences are listed in decreasing level of induction of the most highly induced member of that operon. Promoters of the non-heat shock genes that were induced by translational misfolding are shown in the lower portion of the figure. Nucleotides that are conserved in RpoH recognition sequences are shown in gray shading.
- Figures 2A-C shows a summary of screening results for 18 Thermatoga maritima proteins with pre-determined expression characteristics.
- the average relative ⁇ - galactosidase activity (Figure 2A), Ni-HRP activity ( Figure 2B), and the resulting solubility scores (Figure 2C) for the 18 J. maritima proteins are shown.
- Expression characteristics for the 18 proteins were previously determined by SDS-PAGE of both soluble and insoluble fractions.
- Figure 3 shows the relative ⁇ -galactosidase activity versus the relative Ni- HRP activity observed after expression of 186 T. maritima proteins in a reporter strain. Classification of each protein as soluble, insoluble, or mixed is based on SDS-PAGE performed on the soluble and insoluble lysates after the screen.
- Figure 4 shows an alignment of the secondary structure predictions and both predicted and identified domains of Rep68. Shown are Chou-Fasman secondary structure predictions of ⁇ -helical and ⁇ -sheet structures aligned with a Kyte-Doolittle plot of hydrophobicity based on the primary sequence of Rep68. Also aligned below are blocks representing the relative size and position of: the full-length Rep68 protein, the three predicted domains of Rep68, and the Rep68 domain identified by screening of randomly generated fragments of the rep68 gene. Solubility scores for the proteins are indicated.
- a cell includes a plurality of cells, including mixtures thereof.
- polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
- the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
- Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
- polynucleotide includes, for example, single-double- stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- a nucleic acid molecule may also comprise modified nucleic acid molecules.
- peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
- the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
- a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short.
- the peptide chain is long (e.g., longer than about 10-20 amino acids), the peptide is commonly called a polypeptide or a protein.
- geneticically modified means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous polynucleotides.
- heterologous also refers to a polynucleotide or polypeptide that is not naturally associated with a particular cell or cellular components.
- a promoter that is heterologous to a particular host cell is not found in a naturally occurring cell of that species.
- a promoter that is heterologous to a particular protein-encoding polynucleotide is not found attached to that particular polynucleotide in a naturally occurring cell.
- the term "recombinant" is sometimes used to refer to nucleic acids that include polynucleotides that are not associated with each other in cells that are unmodified by recombinant methods.
- expression refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and translation initiation sequences for ribosome binding.
- a bacterial expression vector includes a promoter such as the lac promoter and for transcription and translation initiation the Shine-Dalgarno sequence and the start codon ATG (Sambrook et al. (2001) supra).
- a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
- a heterologous or homologous promoter for RNA polymerase II for RNA polymerase II
- a downstream polyadenylation signal for RNA polymerase II
- the start codon AUG a downstream polyadenylation signal
- a termination codon for detachment of the ribosome.
- a “promoter” is a region on a DNA molecule to which an RNA polymerase binds and initiates transcription.
- the nucleotide sequence of the promoter determines both the nature of the enzyme that attaches to it and the rate of RINA synthesis.
- the term "promoter” is used to mean a polynucleotide that includes not only the RNA polymerase binding site but also all other contiguous sequence elements that interact with factors which modulate transcription initiation, such as repressors or inducers of transcription.
- a “promoter” as defined here is a polynucleotide that contains all of the sequence information required to regulate gene expression in the same way as the native element in the chromosome.
- protein solubility responsive promoter means a promoter element that is either induced or repressed in a cell in response to an increased concentration of insoluble protein in the cytoplasm.
- "Under transcriptional control” is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. "Operatively linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
- expression construct means a polynucleotide comprising a promoter element operatively linked to a gene.
- the expression construct can be formatted in a variety of ways such as in a gene delivery vehicle or inserted into a chromosome of a cell.
- the term is intended to refer to promoter-gene fusions produced by any method including, but not limited to recombinant DNA techniques, homologous recombination, targeted insertion of a gene or promoter element or random insertion of a gene or promoter element.
- a "gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
- liposomes examples include liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
- Gene delivery ' “gene transfer,” and the like as used herein, are terms refemng to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction.
- exogenous polynucleotide sometimes referred to as a "transgene”
- transgene an exogenous polynucleotide
- Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides).
- the introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
- a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
- a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
- a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
- viral vectors include retroviral vectors, adenovirus vectors, adeno- associated virus vectors and the like.
- a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
- retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
- the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
- retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
- Retro viruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
- the integrated DNA form is called a provirus.
- a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.
- Ads adenoviruses
- Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., WO 95/27071. Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad-derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, WO 95/00655 and WO 95/11984.
- Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Hermonat and Muzyczka (1984) Proc. Nat'l. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.
- Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
- Gene delivery vehicles also include several non-viral vectors, including DNA/liposome complexes, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
- the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
- a reporter gene is a polynucleotide encoding a protein whose expression by a cell can be detected and quantified.
- a measurement of the level of expression of the reporter is indicative of the level of activation of the promoter element that directs expression of the reporter gene.
- detection includes, for example, selection for the presence of reporter gene expression by placing cells that contain the reporter gene under selective conditions.
- Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
- the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
- the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
- a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
- Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6 X SSC to about 10 X SSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 6 X SSC.
- Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9 X SSC to about 2 X SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5 X SSC to about 2 X SSC.
- high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1 X SSC to about 0.1 X SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1 X SSC, 0.1 X SSC, or deionized water.
- hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
- SSC is 0.15 M NaCI and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
- a polynucleotide or polynucleotide region has a certain percentage (for example, 80%, 85%, 90%, or 95%,) of
- sequence identity to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
- This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1.
- a preferred program for aligning polynucleotide and polypeptide sequences to determine percent homology is CLUSTALW, using default parameters.
- a “biological equivalent" of a reference polynucleotide is one characterized by possessing at least 75%, or at least 80%, or at least 90% or at least 95% sequence identity as determined using a sequence alignment program under default parameters, correcting for ambiguities in the sequence data and changes in nucleotide sequence that do not alter function.
- a “biologically equivalent” polynucleotide can also be isolated by hybridization under moderate or stringent hybridization conditions. In addition to sequence similarity or hybridization with reference polynucleotides, the biologically equivalent polynucleotide has the same or similar biological function as the reference polynucleotide.
- BLAST family programs including BLASTN, BLASTP, BLASTX, TBLASTN, and TBLASTX (BLAST is available from the worldwide web at http://www.ncbi.nlm.nih.gov/BLASTI), FastA, Compare, DotPlot, BestFit, GAP, FrameAlign, ClustalW, and PileUp.
- BLASTN BLASTP
- BLASTX BLAST is available from the worldwide web at http://www.ncbi.nlm.nih.gov/BLASTI
- FastA Compare
- DotPlot BestFit
- GAP FrameAlign
- ClustalW ClustalW
- PileUp PileUp
- sequence analysis and alignment programs can be accessed through the world wide web at sites such as the CMS Molecular Biology Resource at www.sdsc.edu/ResTools/cmshp.html.
- Any sequence database that contains DNA or protein sequences corresponding to a gene or a segment thereof can be used for sequence analysis.
- Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ,
- PDB PDB, SWISS-PROT, EST, STS, GSS, and HTGS. Sequence similarity can be discerned by aligning the tag sequence against a DNA sequence database. Alternatively, the tag sequence can be translated into six reading frames; the predicted peptide sequences of all possible reading frames are then compared to individual sequences stored in a protein database such as s done using the BLASTX program.
- Parameters for determining the extent of homology set forth by one or more of the aforementioned alignment programs are well established in the art. They include but are not limited to p value, percent sequence identity and the percent sequence similarity. P value is the probability that the alignment is produced by chance. For a single alignment, the p value can be calculated according to Kariin et al.
- the p value can be calculated using a heuristic approach such as the one programmed in BLAST.
- Percent sequence identify is defined by the ratio of the number of nucleotide or amino acid matches between the query sequence and the known sequence when the two are optimally aligned. The percent sequence similarity is calculated in the same way as percent identity except one scores amino acids that are different but similar as positive when calculating the percent similarity.
- In vivo gene delivery, gene transfer, gene therapy and the like as used herein, are terms referring to the introduction of a vector comprising an exogenous polynucleotide directly into the body of an organism, such as a human or non-human mammal, whereby the exogenous polynucleotide is introduced to a cell of such organism in vivo.
- isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature.
- an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome.
- a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
- a "concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than “concentrated” or less than “separated” than that of its naturally occurring counterpart.
- a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide.
- a protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eucaryotic cell in which it is produced in nature.
- “Host cell,” or “genetically modified cell” are intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in mo ⁇ hology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
- the cells may be procaryotic or eucaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.
- a "subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
- a "control” is an alternative subject or sample used in an experiment for comparison pu ⁇ ose.
- a control can be "positive” or “negative.”
- the pu ⁇ ose of the experiment is to determine a correlation of an altered expression level of a gene with a particular type of cancer, it is generally preferable to use a positive control (a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that disease), and a negative control (a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease).
- the term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (mo ⁇ hologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
- a “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
- a "pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
- the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
- the compositions also can include stabilizers and preservatives.
- stabilizers and adjuvants see Martin REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
- an “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.
- Solid growth media is growth media appropriate to the organism being cultured which contains agar at sufficient concentration to provide a solid surface for the pu ⁇ ose of plating cultures for clonal populations of cells.
- Indicator dyes refer to chemicals which react with the product of the reporter gene to produce a compound with altered properties that can easily be assayed.
- An example of a suitable indicator dye is X-gal which reacts with beta- gal actosidase, the gene product of the lacZ reporter, to produce a blue precipitate.
- the invention provides solubility reporter gene constructs that allow one to readily distinguish whether a protein is produced by a cell in an insoluble form or a soluble form. Also provided are reporter host cells for use in identifying proteins or protein domains that are produced in soluble form, as well as methods for determining the protein solubility state in a cell. In further embodiments, the invention provides high-throughput methods for determining the solubility state of a target protein that is expressed in a cell. Solubility Reporter Gene Constructs
- This invention provides host cells that contain solubility reporter constructs that include a promoter that is induced or repressed depending upon whether insoluble proteins are present in a cell that contains the promoter.
- These protein solubility responsive promoters are preferably linked to a polynucleotide that encodes a gene product that is readily detectable when expressed in a cell.
- a solubility reporter gene construct that includes a promoter that is upregulated by insoluble proteins is present in a cell, for example, the presence of insoluble protein will result in an increase in the level of the reporter gene product.
- Suitable promoters for use in a particular species one can compare gene expression profiles from cells of that species that express a protein that is known to be expressed in an insoluble form to cells that do not express an insoluble protein.
- the control cells can express a protein that is found in soluble form.
- a region upstream of that gene can be cloned and used to construct a solubility reporter construct. The length of the polynucleotide that includes upstream region will sometimes vary depending upon the particular gene and/or species.
- an upstream region is cloned, one can readily test its functionality by operably linking the upstream region to a reporter structural gene, introducing the construct into a host cell, and expressing a protein that is known to be expressed in insoluble form.
- Promoter sequences responsive to misfolded protein can be identified by, for example, Affymetrix GeneChip ® , cDNA array, reporter screening, and other approaches that are known to those of skill in the art.
- the protein solubility responsive promoter can be a prokaryotic or a eukaryotic promoter.
- a promoter that is functional in the particular host cell of interest is utilized.
- Gram negative bacteria include, for example, members of the family Enterobacteriaceae. Examples of the members of the Enterobacteriaceae are the genera Escherichia, Salmonella, Shigella, Klebsiella or Enterobacter.
- Suitable prokaryotic cells include, but are not limited to
- E. coli Salmonella typhomurium, Bacillus subtilis and Streptomyces lividans.
- One suitable species is a promoter element isolated from the Gram negative bacterium E. coli.
- E. coli promoters include, for example, promoters from the following genes: kgi? gene (b2587; S ⁇ Q ID NO: 1), gene b3913 (S ⁇ Q ID NO:2), pro?
- the protein solubility responsive promoters include an RpoH recognition site. Examples of such promoters are shown in Figure 1, and as SEQ ID NOS.23-43.
- This invention also encompasses the use of biologically equivalent polynucleotides to the sequences provided in Seq. ID. Nos. 1-43, which can be identified using sequence homology searches or hybridization under moderate or tringent hybridization conditions as defined above.
- biologically equivalent polynucleotides are within the scope of this invention, e.g., those characterized by possessing at least 75%, or at least 80%, or at least 90% or at least 95% sequence homology as determined using a sequence alignment program under default parameters correcting for ambiguities in the sequence data, and changes in nucleotide sequence that do not alter function.
- Biological equivalents also includes those that hybridize under conditions of moderate or stringent conditions to the sequences of Seq. ID. Nos. 1-43, or their respective complements. Such polynucleotides can be tested according to the methods of the invention to identify those that exhibit the desired protein solubility responsiveness.
- a protein solubility responsive promoter is generally obtained from a eukaryotic gene.
- Many eukaryotic heat shock and other stress- induced genes are known to those of skill in the art.
- the invention provides methods for testing promoters from these and other genes to determine whether the promoters are differentially regulated in response to the presence of an insoluble protein in the cell. These methods involve culturing a host cell that includes a solubility reporter nucleic acid that comprises a putative protein solubility responsive promoter operably linked to a reporter gene.
- the host cell also contains a target polypeptide-expressing nucleic acid that includes a polynucleotide that encodes a target polypeptide.
- the host cell is cultured under conditions in which the target polypeptide is expressed in insoluble form.
- the level of expression of the reporter gene is then detected to determine whether the putative protein solubility responsive promoter is differentially regulated in response to expression of an insoluble polypeptide in the host cell.
- Suitable eukaryotic cells include, for example, mammalian, insect, or plant cells or microorganisms, such as, for example, yeast cells, or fungal cells.
- suitable cells include, for example, Azotobacter sp. (e.g., A. vinelandii), Pseudomonas sp., Rhizobium sp., Erwinia sp., Escherichia sp. (e.g., E. coli), and Klebsiella sp., among many others.
- Yeast cells can be of any of several genera, including Saccharomyces (e.g., S. cerevisiae), Candida (e.g., C. utilis, C.
- parapsilosis C. krusei, C. versatilis, C. lipolytica, C. zeylanoides, C. guilliermondii, C. albicans, and C. humicola
- Pichia e.g., P.farinosa and P. ohmeri
- Torulopsis e.g., T. Candida, T. sphaerica, T. xylinus, T. famata, and T. versatilis
- Debaryomyces e.g., D. subglobosus, D. cantarellii, D. globosus, D. hansenii, and D.
- Suitable eukaryotic cells include Jurkat cells and NIH3T3 cells.
- the protein solubility responsive promoters identified above are operatively linked to a reporter gene that functions to identify the presence or absence of soluble protein in the cell cytoplasm.
- the reporter genes include a polynucleotide that encodes a selectable or detectable polypeptide. Examples of genes useful as "reporter genes" include, but are not limited genes that encode a metabolic enzyme, an antibiotic resistance factor, a luminescent protein (e.g., luciferase), or a fluorescent protein. Such reporter genes are well known in the art and particular examples are described in Wood (1995) Curr. Opin. Biotechnol. 6(I):50-58.
- the metabolic enzyme is ⁇ -galactosidase.
- the metabolic gene is a gene that complements an auxotrophic mutation in a host cell and allows growth of cells that express the gene on selective media.
- Methods for detecting and quantitating reporter expression are commonly based on measuring the activity of the protein encoded by the reporter.
- detectable markers include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
- enzyme tags colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
- the reporter is an enzyme
- a substrate for the enzyme which is metabolized to produce a measurable product can be used.
- the ⁇ -galactosidase substrate X-gal which is cleaved by this enzyme to produce a blue reaction product, is frequently used to assay ⁇ -galactosidase reporter expression.
- the ⁇ -galactosidase substrate o-nitrophyl-B-D-galactopyranoside (ONPG), which is metabolized by ⁇ -galactosidase to produce a compound with a yellow color.
- the quantity of enzyme is determined by measuring optical density of the colored compound spectrophotometrically or with an ELISA reader. The absorbance is read at 420nm (Miller J.H. ed. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
- reporter genes are the antibiotic resistance factor chloramphenicol acetyl transferase (CAT), the firefly luciferase gene, and the jellyfish green fluorescent protein (Valdivia and Falkow (1997) Trends Microbiol. 5(9):360-363; Naylor (1999) Biochem. Pharmacol. 58(5):749-757; Himes and Shannon (2000) Methods Mol. Biol. 130:165-174).
- CAT antibiotic resistance factor chloramphenicol acetyl transferase
- the firefly luciferase gene the firefly luciferase gene
- the jellyfish green fluorescent protein a variety of alternative proteins can also be used as reporters based on their ability to be detected and quantitated. Assays to measure the expression levels of such genes are well developed and are commonly practiced by those of ordinary skill (Rosenthal (1987) Methods Enzymology 152:704-720; Davey et al.
- Polynucleotides that encode useful reporter genes are available from a variety of commercial suppliers of molecular biology reagents such as LifeTechnologies Inc. (Gaithersburg, MD), Clontech Inc. (Palo Alto, CA), Promega Inc. (Madison, WI), Invitrogen Inc. (Carlsbad, CA), and Strategene Inc. (San Diego, CA).
- plasmid vectors comprising reporter gene sequences are available from the American Type Culture
- the solubility reporter nucleic acids of the invention can comprise additional sequences, such as coding sequences within the same transcription unit, controlling elements such as ribosome binding sites, and polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
- the solubility reporter nucleic acids include a polynucleotide that encodes a signal peptide that directs a detectable polypeptide encoded by the reporter gene to a surface of the host cell. The detectable polypeptide can then be detected by, e.g., a cell sorter. For example, if the reporter gene encodes a fluorescent protein, which is displayed on the surface of the cell upon expression, one can utilize a fluorescence activated cell sorter to separate cells that express the reporter gene from those that do not.
- the solubility reporter nucleic acids can also include a polynucleotide that encodes a molecular tag which can facilitate separation of a host cell that expresses the reporter gene from a host cell that does not express the reporter gene.
- a molecular tag which can facilitate separation of a host cell that expresses the reporter gene from a host cell that does not express the reporter gene.
- an epitope for an antibody can function as a molecular tag; cells that express the reporter gene can then be immobilized by contacting the cells with a solid support to which is attached antibodies that specifically recognize the epitope.
- Other suitable molecular tags are well known to those of skill in the art, and include, for example, a poly-histidine tag, or a FLAGTM peptide.
- the invention also provides a reporter system comprising: a) an isolated polynucleotide containing at least a protein solubility responsive promoter operatively linked to a reporter gene, and b) an expression construct that directs the expression of a target gene.
- the expression construct can be either on a separate polynucleotide from the promoter and reporter gene or the expression construct can be part of a single polynucleotide that also contains the protein solubility responsive promoter and reporter gene.
- the reporter system comprises an isolated polynucleotide with a protein solubility responsive promoter operatively linked to a reporter gene, wherein the isolated polynucleotide further comprises an expression construct.
- the present invention also provides gene delivery vehicles suitable for delivery and/or expression of a polynucleotide of the invention into cells (whether in vivo, ex vivo, or in vitro) containing the polynucleotides of this invention.
- a polynucleotide of the invention can be contained within a cloning or expression vector.
- These vectors (especially expression vectors) can in turn be manipulated to assume any of a number of forms which may, for example, facilitate delivery to and/or entry into a cell. Examples of suitable expression and delivery vehicles are provided above.
- This invention also provides host or genetically modified cells containing the protein solubility reporter constructs described above, as well as a target polypeptide- expressing nucleic acid that includes a polynucleotide that encodes a target polypeptide identified above.
- Arrays of cells are also provided, in which the cells of each population differ in the target polypeptides expressed by the cells.
- the polypeptides can differ due to amino acid substitutions, deletions, or insertions compared to a reference amino acid sequence.
- the target polypeptides expressed by the populations of host cells can be different fragments of a larger polypeptide.
- the polynucleotides and sequences embodied in this invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof.
- the PCR technology is the subject matter of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: THE POLYMERASE CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995), supra, and references cited therein.
- one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA.
- this invention also provides a process for obtaining the polynucleotides of this invention by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides.
- these polynucleotides are further isolated.
- one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification.
- the DNA so amplified can be isolated from the cell by methods well known to those of skill in the art.
- a process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained.
- RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell.
- the DNA can be inserted by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation.
- an appropriate gene delivery vehicle e.g., liposome, plasmid or vector
- electroporation e.g., liposome, plasmid or vector
- the RNA can then be isolated using methods well known to those of skill in the art, for example, as set forth in Sambrook et al. (2001) supra.
- mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al.
- compositions containing a carrier and the polynucleotides and sequences of this invention, in isolated form or contained within a vector or host or genetically modified cell are further provided herein. When these compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.
- polynucleotides, reporter systems and cells are useful in the methods described below.
- a cell containing a construct of this invention is cultured under conditions where the target protein is expressed and the expression of the reporter gene is inducible.
- the term "inducible” shall mean that transcription of the reporter gene can be initiated in response to a specific stimulus.
- the specific stimulus that induces transcription of a protein solubility responsive promoter is insoluble protein in the cytoplasm of the cell.
- the cells of the Gram negative bacterium E. coli for example, the cells should be grown in liquid medium rather than on agar plates for the reporter gene to be inducible.
- Expression of the reporter gene is measured following expression of the target protein. This can be accomplished by measuring the amount of protein directly such as by measuring fluorescence of a fluorescent protein or by measuring the reporter protein by an immunoassay such as an ELISA assay. Alternatively, if the reporter gene is an enzyme, the amount of reporter produced can be measured using an assay that quantifies a product produced by enzymatic modification of a substrate compound, such as metabolism of X-gal or ONPG by the ⁇ -galactosidase enzyme. The amount of reporter protein produced will be directly proportional to the amount of insoluble target protein in the cytoplasm.
- the quantity of insoluble protein in a specific sample can be determined by first preparing a standard curve correlating target protein insolubility with the level of reporter gene expression. This can be accomplished by culturing a host cell comprising the reporter construct together with a target expression construct and preparing a series of samples in which the various amounts of insoluble target protein are produced. Expression of the protein insolubility reporter is measured in each of these samples. [0081] The amount of soluble and insoluble target protein can be measured quantitatively by lysing the host cells, separating soluble and insoluble material, for example by centrifugation or filtration, and measuring the amount of target protein in each fraction, for example by immunoassay such as ELISA or Western blot.
- the amount of insoluble protein present in a test sample can be determined by measuring the expression of the protein insolubility reporter in that sample and calculating the amount of insoluble protein present from the standard curve.
- the invention also provides a method of screening for mutations in a cell that improve the solubility of a protein. These methods involve treating a population of cells with a mutagen, and identifying those cells that exhibit an increase in expression of the target protein in soluble form.
- a "mutagen” is intended to include, but not be limited to chemical mutagens such as ethyl methane sulphonate, N-methyl-N'-nitroso-guanidine and nitrous acid as well as physical agents such as ionizing radiation.
- mutations can be introduced into a polynucleotide sequence encoding a target protein. The altered polynucleotide is then tested to determine whether the solubility of the target protein is changed.
- Such mutations include for example, mutations induced by a mutagen; site directed mutations that alter specific amino acid residues such as mutation of cysteine residues to eliminate disulfide bonds; deletions that remove sets of specific amino acids such as deletion of a continuous stretch of hydrophobic amino acids; and fusions of the target protein to a second, particularly soluble protein.
- the solubility of the target protein is assessed by determining expression of a protein solubility reporter nucleic acid as described herein.
- a polynucleotide that encodes this protein is expressed suitable conditions such that the reporter gene is responsive to expression of insoluble protein. If a mutation has been introduced that increases the solubility of the target protein then the level of expression of the reporter gene will be reduced as compared to the level of expression of the reporter gene observed in the host cell prior to treatment of this cell with the mutagen, provided that the protein solubility responsive promoter is upregulated in response to expression of insoluble protein.
- the constructs are also useful for identifying variations in a process for biosynthesis of a target protein.
- the process can be varied to modify the solubility of the target protein.
- a cell containing a protein solubility reporter nucleic acid is cultured under alternative conditions where the target protein is expressed and the reporter is inducible, and measuring the expression of the reporter gene, to identify variations in culture conditions that improve the solubility of the expressed target protein.
- protein solubility may be affected by the temperature, medium composition, or oxygen concentration in which the cells are cultured.
- the convenient method by which expression of the reporter is measured allows a variety of alternative conditions to be tested with minimal effort, to identify those conditions where the highest proportion of soluble target protein is produced.
- constructs also are useful to compare alternative cells to identify a cell that synthesizes an increased amount of soluble target protein by performing a method identified herein with at least two alternative cells and comparing the amount of reporter gene expressed to identify a cell that expresses an increased amount of soluble target protein.
- the present invention also provides a method of screening an expression library of clones to identify those clones that express soluble protein.
- This library can consist of alterations in the gene expressing the target protein of interest. Alterations of the gene can be provided by any of several widely used methods. These include making truncations in the gene, random chemical mutagenesis, random mutagenesis through erroneous nucleotide inco ⁇ oration, or site-directed mutagenesis methods.
- This library of alterations is transformed into cells that contain the protein solubility reporter system. Individual clones of the transformed cells are then cultured under conditions where the target gene or its alterations are expressed. The level of reporter gene expression in each clone is measured during expression of target gene or its alterations.
- Clones expressing increased or reduced levels of the reporter gene are identified by measuring reporter gene levels of each clone and comparing to a clone expressing the unmodified target gene. Clones thus identified are expressing less insoluble protein and may contain more soluble derivatives of the target protein.
- reporter genes for the protein solubility reporter system will enable the use of this system in a variety of efficient, high-throughput procedures to rapidly screen large number of alternative cultures in order to identify specific samples that produce soluble target proteins.
- reporter genes such as ⁇ -galactosidase, luciferase, and green fluorescent protein further provides for the development of automated procedures to screen cells for target protein solubility.
- the constructs as defined herein are useful for identifying an antibiotic agent.
- the cells that contain the protein solubility reporter construct are contacted by a candidate agent.
- a potential antibiotic agent that interferes with the protein folding process will result in an increased expression of insoluble endogenous cellular protein, thereby inducing expression of a reporter gene that is under the control of a promoter that is upregulated in response to the presence of insoluble protein.
- Measurement of the reporter gene product is performed after treatment with the potential antibiotic agent.
- Cells expressing increased reporter activity relative to a control substance are an indication that the test agent is a potential antibiotic.
- An additional aspect of the invention is to use the process described above with co-expression of a soluble protein which is a known target of antibiotic therapies. Agents that interfere with the folding of these known target proteins would result in insoluble protein and increased expression of the reporter gene. Agents thus identified would have potential utility as an antibiotic by interfering with the proper folding of these target proteins in their native hosts.
- Clones expressing properly folded or misfolded human proteins were obtained from the GeneStorm collection (Invitrogen). Clones containing the Unigene accession numbers L35545, U18291, M94856, M22146, D871 I6, M63167, M68520, M60527, M36881, M36981, U35003, S79522, X73460, D14520, U14968, M86400 were provided in the pBADThio vector (Invitrogen) to provide arabinose-inducible expression. T.
- maritima genes were amplified from genomic DNA and cloned into the expression vector pMHl which encodes a 12 amino acid N-terminal tag containing a 6X-histidine repeat for purification and detection.
- Reporter vectors were constructed by inserting a PCR amplifer of 300 bp upstream of the ibpAB, ybeD, yhgl or yrfG ⁇ l genes upstream of beta-galactosidase in a pACYC184 derivative.
- Rep 68 was cloned from a plasmid that contains the entire genome of the human adeno-associated virus 2 (AAV2).
- Putative domains comprised of bases 1-646, 647- 1456, and 1457-1611 were amplified from the full-length template and cloned into pMHl.
- the above template was also used in amplifications of the full-length gene for fragmentation.
- Two ⁇ g of the rep 68 amplifer were used in each of 5 fragmentation reactions containing 1, 0.1, 0.01, 0.001, or 0 units of DNase I (Boeringer Mannheim) as well as Pfu polymerase and dNTPs. Reactions were set up on ice with the DNase added immediately prior to temperature cycling in an MJ Research thermocycler according to the following: 10 min @ 25°C, 15 min @ 95°C, and 30 min @ 72°C.
- Each reaction was run on a 1% agarose gel and fragments corresponding to 1600-1000 bp, 1000-850 bp, 850-600 bp, and 600-300 bp were extracted. Each pool was used as above for blunt cloning and ligation into pMHl as above and introduced into the reporter cell line HK 57 for screening.
- E. coli strains MG1655 (F lam rph ) and KY1429 (F- ⁇ raD139 A(argF- lac)169 lamflhD5301 fruA25 relAl rpsL150 zhh50::TnlO rpoR606(ts) deoCl) were transformed with expression plasmids encoding M36881 (LCK) or M86400 (PLA) for expression profiling.
- LCK LCK
- PHA M86400
- KY1429 cells were cultured as above except initial growth was performed at 32° C followed by a shift to 42°C for non-permissive expression of rp ⁇ H606.
- Top 10 cells F mcrA A(mrr-hsdRMS-mcrBC) ⁇ 80/ ⁇ cZ ⁇ M15 ⁇ / ⁇ cX74 deoR recAl ⁇ r ⁇ D139 A(ara- leu)l691 ga ⁇ U galK rpsL endA nupG) containing the ibp AB promoter fusion (pHK57), were transformed with expression constructs listed above.
- Beta-galactosidase assays were performed essentially as described by Miller (24). Fractionation of soluble and insoluble proteins was performed by centrifugation.
- Probe preparation and hybridization and analysis of labeled mRNA [0095] Labeled mRNA was prepared and hybridized to an E. coli whole genome array (Affymetrix) essentially as described previously (25, 26). This gene chip contains 25- mer oligonucleotide probes for each of the 4290 known E. coli genes. Standard Affymetrix GeneChip analysis software was used to measure individual gene expression and to perform pairwise comparison of gene expression levels for pre-induction and post-induction samples. Comparisons of changes in gene expression for properly folded and misfolded genes were analyzed for individual gene probe sets.
- Affymetrix E. coli whole genome array
- Microplate Solubility Screening Ninety-six well microplates containing 200 ⁇ L of LB with 100 ⁇ g/mL ampicillin and 34 ⁇ g/mL chloramphenicol were inoculated with single colonies from above and grown overnight with shaking at 37°C. Overnight cultures were used to inoculate 200 ⁇ L of the same media and incubated at 37°C until reaching an average OD 6 oo of 0.5. Cultures were induced with a final concentration of 0.2% arabinose. After 30 minutes, a cocktail of cefatriaxone and cefotaxime was added to each well to a final concentration of 10 ⁇ g/mL of each and the plates were incubated for an additional 1.5 hours.
- Cultures were harvested after 2 hours total of induction by centrifugation at max speed for 15 minutes to pellet cell debris on the bottom of the wells.
- the soluble lysate was then separated 25 ⁇ L into one set of clean microplates for ⁇ -galactosidase activity screens and 75 ⁇ L into Nunc Maxiso ⁇ TM ⁇ LISA plates for Ni-HRP screening.
- ⁇ -galactosidase activity screening of lysates was performed using a variation of the Miller protocol (10). 50 ⁇ L of 4x Z-buffer and 50 ⁇ L of 4x ONPG were added to microplates containing 25 ⁇ L of soluble lysate. After development of yellow color in positive control wells, the reaction was quenched with 75 ⁇ L of 1M Na 2 CO 3 pH 8. The A 420 , A 550 and reaction times were recorded and used along with the OD 6 oo data to calculate ⁇ -galactosidase activity (10). [0098] Ni-HRP screening was performed similar to an ELISA.
- Solubility scores were calculated by weighting the Ni-HRP A 20 readings such that the mean was one order of magnitude greater than the mean of the ⁇ -galactosidase activity scores and dividing the Ni- HRP absorbance by the ⁇ -galactosidase activity.
- mRNA preparations from induced and non-induced cultures were prepared and used to probe for gene expression.
- Recombinant protein expression within E. coli is predicted to cause a substantial change in gene expression. Indeed, a comparison of gene expression with a pre- induction control shows 6% of total genes showing >3-fold differences in expression in both cases. In the case of insoluble recombinant protein, 27 genes show >10-fold changes in expression, as compared to 10 genes in the case of the soluble recombinant protein. A comparison of the two profiles identifies 53 genes listed in Table 1 showing >3-fold changes, that are unique to the insoluble case. These genes, then, are likely responsive to misfolded protein in the cell and may play a role within E. coli in dealing with this translational stress.
- the heat shock transcription factor RpoH is normally repressed by interaction with the chaperone protein DnaK. In the presence of misfolded protein, DnaK binds to that protein thereby allowing RpoH to stimulate transcription of heat shock promoters (7). Upstream regions of many of the induced genes in Table 1 show the presence of RpoH-dependant promoter sequences. Further evidence of the important role played by RpoH is provided by expression profiling results performed from an rpoH606 mutant (KYI 429) expressing misfolded LCK protein compared to a non-expressing control. A strikingly different expression profile is seen in the case of the oH606 mutant (Tables 1 & 2). The majority of the genes induced by the misfolded protein in the wild-type strain are poorly induced in the oH606 mutant indicating that they are directly or indirectly under control of this transcription factor.
- Hsp33 the gene product of the yrf gene was recently identified as a chaperone protein responsive to oxidizing conditions (15). Genes implicated in degradation of denatured protein are also induced by translational misfolding. The Ion, clpB?, and hslUN protease genes are expressed at increased levels. Under normal cell growth these proteases serve an important recycling function. Insoluble aggregates are relatively resistant to proteolysis and this recycling pathway is ineffective for recombinant protein expression. Table 1. Fold change in gene expression for genes unique to misfolded response.
- ribosomal dissociation comes from the induction of ftsJ (rrrr ⁇ ) (SEQ ID NO: 10).
- The/ttJ gene product is an RNA methylase specific for 23S rRNA only when contained in the 50S ribosomal subunit (17, 18). This enzyme methylates 23S rRNA at position 2552 located within the peptidyl transferase center of the ribosome (17).
- Mutants infisJ lack methylation of 23S rRNA and show up to 65% decrease in ribosomal activity corresponding to dissociation of the 50S and 30S subunits (19).
- CSPs cold-shock proteins
- yccV, yhdN, and yrfG have been shown to increase expression under heat shock conditions but are of unknown function.
- yagU, yciS, ybeD, yejG, and yhgl show increased expression.
- Most of these proteins are relatively small and generally acidic.
- IbpAB perform a similar role to IbpAB in the direct recognition and sequestering of misfolded protein.
- IbpAB have been associated with misfolded and aggregated protein. Induction levels of ibpAB are much higher and these other proteins may be present at lower levels.
- a negative Ni-HRP response therefore, may not be indicative of an absence of soluble protein, but the protein fold may occlude access to the His-tag.
- This assay provides a measure of the levels of soluble recombinant protein without the need to run an SDS gel and in a form that is compatible with a HT-screen and the ⁇ -galactosidase assay. Testing Proteins with Pre-determined Expression Characteristics [0107] As part of our effort aimed at cloning, expressing, and characterizing the total proteome of Thermotoga maritima, we tested the efficacy of the reporter on a set of 18 T.
- maritima proteins shown in Table 3 (6 soluble, 6 insoluble, and 6 mixed solubility).
- Figure 2 shows the averaged results for triplicate plates (soluble, insoluble and mixed) for the 0.2% arabinose induction. Both the insoluble and the mixed pools showed greater than four-fold higher ⁇ -galactosidase activity than the soluble pool ( Figure 2A).
- the soluble pool showed a greater than ten-fold higher response in the Ni-HRP assay opposed to the insoluble pool (Figure 2B).
- the mixed pool comprised of proteins expressed approximately equally in both soluble and insoluble fractions, showed Ni-HRP binding approximately half the intensity of the soluble pool.
- Table 3 T. maritima proteins with pre-determined expression characteristics Accession # ID MW pj
- Soluble Expression TM0560 conserved hypothetical protein 20.62 5.34
- TM0554 3-isopropylmalate dehydratase, large subunit (leuC) 45.286 5.92
- TM0556 3-isopropylmalate dehydrogenase (leuB) 39.19 5.44
- FIG. 3 A comparison of ⁇ - galactosidase activity to Ni-HRP assay is shown in Figure 3. Points are categorized by SDS gel analysis of the soluble and insoluble protein fractions. The screen positively identified 54 of 62 (87%) soluble proteins. Seven of the eight remaining proteins that were soluble according to the gels had low Ni-HRP assays, most likely due to inaccessibility of the His- tag in these fusion proteins. Taken alone, the ⁇ -galactosidase activity measurement identified 22 of 27 (81%) insoluble proteins. Those proteins showing partial solubility showed variable solubility scores, suggesting partial folding is inducing ⁇ -galactosidase through the reporter. This assay, then, provides an effective and convenient means of classifying folding characteristics.
- Ribosome structure shows that the location of the methylation site of fisl, position 2552 of the 23S rRNA, is intriguingly close to the peptidyl transferase center (18) making it an obvious potential regulator mechanism for a ribosomal sensor of misfolded protein.
- a ribosomal sensor is not unprecedented as demonstrated by the well-characterized stringent response to uncharged tRNAs during translation (23).
- Ribosomal stalling provides a mechanism to allow time for chaperone synthesis and recruitment thereby preventing irreversible aggregation. In this way, the cell would retain an additional salvage pathway where the emerging protein was held in the relatively protected environment of the translating ribosome until sufficient chaperones could be recruited.
- the differentially regulated genes identified provide a valuable opportunity to create novel reporters of the folding state of cellular proteins as a whole and over-expressed, recombinant proteins in particular.
- Our reporter assay differs from others recently described by not relying on direct coupling of the reporter gene to the target, thereby limiting potential interference by the reporter.
- the combination of the Ni-HRP and ⁇ galactosidase assays provides an effective means of assaying soluble recombinant proteins in a high-throughput way. We have extended this system to identify mutants and truncations of single gene products as a strategy to identify soluble domains of otherwise misfolded, aggregated proteins. Using this approach, we have identified soluble fragments of Rep68 and anticipate that this assay will provide a general means of isolating recombinant protein suitable for structure/function work. References
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01998134A EP1364055A4 (en) | 2000-11-21 | 2001-11-21 | Solubility reporter gene constructs |
CA002428248A CA2428248A1 (en) | 2000-11-21 | 2001-11-21 | Solubility reporter gene constructs |
JP2002561598A JP2004535772A (en) | 2000-11-21 | 2001-11-21 | Soluble reporter gene structure |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US72134000A | 2000-11-21 | 2000-11-21 | |
US09/721,340 | 2000-11-21 | ||
US32483301P | 2001-09-24 | 2001-09-24 | |
US60/324,833 | 2001-09-24 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2002061041A2 true WO2002061041A2 (en) | 2002-08-08 |
WO2002061041A3 WO2002061041A3 (en) | 2003-09-04 |
WO2002061041A9 WO2002061041A9 (en) | 2004-05-13 |
Family
ID=26984650
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2001/051426 WO2002061041A2 (en) | 2000-11-21 | 2001-11-21 | Solubility reporter gene constructs |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1364055A4 (en) |
JP (1) | JP2004535772A (en) |
CA (1) | CA2428248A1 (en) |
WO (1) | WO2002061041A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016062819A1 (en) * | 2014-10-22 | 2016-04-28 | Danmarks Tekniske Universitet | A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109055417B (en) * | 2018-08-28 | 2020-07-07 | 浙江新和成股份有限公司 | Recombinant microorganism, preparation method thereof and application thereof in production of coenzyme Q10 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5589337A (en) * | 1992-07-06 | 1996-12-31 | The President And Fellows Of Harvard College | Methods and diagnostic kits for determining toxicity utilizing bacterial stress promoters fused to reporter genes |
US5731163A (en) * | 1994-11-23 | 1998-03-24 | E. I. Du Pont De Nemours And Company | Lyophilized bioluminescent bacterial reagent for the detection of toxicants |
-
2001
- 2001-11-21 EP EP01998134A patent/EP1364055A4/en not_active Withdrawn
- 2001-11-21 CA CA002428248A patent/CA2428248A1/en not_active Abandoned
- 2001-11-21 WO PCT/US2001/051426 patent/WO2002061041A2/en not_active Application Discontinuation
- 2001-11-21 JP JP2002561598A patent/JP2004535772A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5589337A (en) * | 1992-07-06 | 1996-12-31 | The President And Fellows Of Harvard College | Methods and diagnostic kits for determining toxicity utilizing bacterial stress promoters fused to reporter genes |
US5731163A (en) * | 1994-11-23 | 1998-03-24 | E. I. Du Pont De Nemours And Company | Lyophilized bioluminescent bacterial reagent for the detection of toxicants |
Non-Patent Citations (5)
Title |
---|
LESLEY ET AL.: 'Gene expression response to misfolded protein as a screen for soluble recombinant protein' PROTEIN ENG. vol. 15, no. 2, 2002, pages 153 - 160, XP002962868 * |
PARSELL ET AL.: 'Induction of a heat shock-like response by unfolded protein in escherichia coli: dependence on protein level not protein degradation' GENES DEV. vol. 3, August 1989, pages 1226 - 1232, XP002962866 * |
See also references of EP1364055A2 * |
TROTTER ET AL.: 'Misfolded proteins are competent to mediate a subset of the responses to heat shock in saccharomyces cerevisiae' J. BIOL. CHEM. vol. 277, no. 47, 22 November 2002, pages 44817 - 44825, XP002962867 * |
WIGLEY ET AL.: 'Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein' NAT. BIOTECHNOL. vol. 19, 19 February 2001, pages 131 - 136, XP002962869 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016062819A1 (en) * | 2014-10-22 | 2016-04-28 | Danmarks Tekniske Universitet | A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation |
US10544414B2 (en) | 2014-10-22 | 2020-01-28 | Danmarks Tekniske Universitet | Two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation |
Also Published As
Publication number | Publication date |
---|---|
JP2004535772A (en) | 2004-12-02 |
WO2002061041A9 (en) | 2004-05-13 |
CA2428248A1 (en) | 2002-08-08 |
EP1364055A4 (en) | 2004-08-25 |
WO2002061041A3 (en) | 2003-09-04 |
EP1364055A2 (en) | 2003-11-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Lesley et al. | Gene expression response to misfolded protein as a screen for soluble recombinant protein | |
Wall et al. | The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication. | |
Jonas et al. | Proteotoxic stress induces a cell-cycle arrest by stimulating Lon to degrade the replication initiator DnaA | |
Yamamoto et al. | Characterization of copper-inducible promoters regulated by CpxA/CpxR in Escherichia coli | |
Bianchi et al. | Stress responses as a tool to detect and characterize the mode of action of antibacterial agents | |
Wendisch et al. | Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays | |
Green et al. | Mutations at nucleotides G2251 and U2585 of 23 S rRNA perturb the peptidyl transferase center of the ribosome | |
Bellier et al. | ClgR, a novel regulator of clp and lon expression in Streptomyces | |
Bergler et al. | Inhibition of lipid biosynthesis induces the expression of the pspA gene | |
Sweasy et al. | Detection and characterization of mammalian DNA polymerase beta mutants by functional complementation in Escherichia coli. | |
Słomińska et al. | Regulation of bacteriophage λ development by guanosine 5′-diphosphate-3′-diphosphate | |
Ziemski et al. | Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms | |
Kuhar et al. | Codon‐usage based regulation of colicin K synthesis by the stress alarmone ppGpp | |
Singh et al. | Recycling of ribosomal complexes stalled at the step of elongation in Escherichia coli | |
US20030119094A1 (en) | Solubility reporter gene constructs | |
EP1364055A2 (en) | Solubility reporter gene constructs | |
AU2002249889A1 (en) | Solubility reporter gene constructs | |
Scocchi et al. | Investigating the mode of action of proline-rich antimicrobial peptides using a genetic approach: a tool to identify new bacterial targets amenable to the design of novel antibiotics | |
CN106591347B (en) | A kind of expression system comprising phage lysozyme and its utilization | |
Simon et al. | Applications of chimeric genes and hybrid proteins, Part A: Gene Expression and Protein Purification | |
Satheeshkumar et al. | Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21 (DE3) is toxic to the host cells | |
Raina et al. | AzuCR RNA modulates carbon metabolism as a dual-function RNA in Escherichia coli | |
van Zyl et al. | Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius | |
US11866712B2 (en) | System based on the reassembly of GFP for studying the trans-translational activity and identifying new antibiotics | |
Babu et al. | Elevated Levels of the Escherichia coli nrdAB-Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the β Clamp |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2428248 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2001998134 Country of ref document: EP Ref document number: 2002561598 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2002249889 Country of ref document: AU |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 2001998134 Country of ref document: EP |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1/4-4/4, DRAWINGS, REPLACED BY NEW PAGES 1/4-4/4; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2001998134 Country of ref document: EP |