WO1999027108A1 - Proteine represseur du lactose a sensibilite du ligand modifiee - Google Patents

Proteine represseur du lactose a sensibilite du ligand modifiee Download PDF

Info

Publication number
WO1999027108A1
WO1999027108A1 PCT/US1998/024949 US9824949W WO9927108A1 WO 1999027108 A1 WO1999027108 A1 WO 1999027108A1 US 9824949 W US9824949 W US 9824949W WO 9927108 A1 WO9927108 A1 WO 9927108A1
Authority
WO
WIPO (PCT)
Prior art keywords
repressor protein
protein
ligand
lac repressor
lac
Prior art date
Application number
PCT/US1998/024949
Other languages
English (en)
Inventor
Kathleen S. Matthews
Liskin Swint-Kruse
Original Assignee
William Marsh Rice University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by William Marsh Rice University filed Critical William Marsh Rice University
Priority to AU14673/99A priority Critical patent/AU1467399A/en
Publication of WO1999027108A1 publication Critical patent/WO1999027108A1/fr
Priority to US10/197,053 priority patent/US7390645B2/en

Links

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to repressor proteins that recognize lactose operator and have altered ligand responsivity.
  • the lac repressor protein is a genetic regulatory protein used widely to control the expression of cloned genes and is the prototype for negative control of transcription initiation in E. coli (Jacob & Monod, 1961). This protein normally regulates expression of the lactose metabolic enzymes and couples cellular response with environmental availability of metabolites (Miller & Reznikoff, 1980). Multiple vector systems are commercially available that employ this protein in cloning genes and overexpressing their protein products.
  • Figure 1 shows a schematic of how the lac repressor protein (Lad) and lac operator (O) work.
  • the i gene product is tetrameric lactose repressor protein (Lad,
  • lactose When lactose is available in the environment, the low constitutive amounts of lac permease transport this sugar into the cell, and the correspondingly low levels of ⁇ - galactosidase result in production of the in vivo inducer, ⁇ -l,6-allolactose.
  • This sugar is the natural inducer of Lad (Jobe & Bourgeois, 1972).
  • lactose levels When lactose levels are decreased, the intracellular store of natural inducer is depleted by ⁇ -galactosidase hydrolysis. These enzymatic activities ensure that the lac enzymes are not expressed except in the presence of lactose.
  • IPTG isopropyl- ⁇ ,D-thiogalactoside
  • ABSP arabinose binding protein
  • HTH refers to the helix-turn-helix domain of lac repressor protein.
  • IPTG refers to isopropyl- ⁇ ,D-thiogalactoside.
  • LHR refers to the leucine heptad repeat domain of lac repressor protein.
  • MMG refers to methyl umbelliferyl- ⁇ ,D-galactoside.
  • X-gal refers to 5-bromo-4-chloro-3-indolyl- ⁇ ,D-galactoside.
  • alternate inducer ligand means a sugar or other small molecule other than allolactose or IPTG, i.e. a sugar or small molecule that is not an inducer ligand of wild-type lac repressor.
  • altered lac repressor protein means a repressor protein which recognizes and can bind to the lac operator sequence, but which has either responsivity to an alternate inducer ligand, or increased affinity for IPTG relative to the affinity of wild- type lac repressor protein for IPTG. Affinity for a ligand is defined as 1/K d , where K d is the concentration of ligand required to occupy 50% of the available ligand binding sites.
  • altered ligand responsivity means either a response to an alternate inducer ligand effective at reasonable concentrations to allow transcription of mRNA or a response to at least 10-fold lower IPTG concentration than wild type repressor.
  • natural lac repressor protein means the repressor protein (La ) naturally found which recognizes and can bind to the lac operator sequence, and which has a responsivity to allolactose or IPTG.
  • substantially homology means reasonable functional and/or structural equivalence between sequences of amino acids or DNA.
  • the present invention provides altered lac repressor proteins comprising a DNA- binding domain of natural lac repressor protein and a ligand binding domain having a responsivity to an alternate inducer ligand or increased sensitivity to IPTG.
  • the altered lac repressor proteins can further comprise a tetramerizing domain of natural lac repressor protein.
  • the alternate inducer ligand is arabinose.
  • the present invention also provides DNA sequences encoding the altered lac repressor proteins and bacterial and eukaryotic cells containing the altered lac repressor proteins.
  • the altered lac repressor proteins of the present invention can be produced by fusing the DNA-binding domain of natural lac repressor protein to the N-terminus of a ligand binding protein followed by mutagenesis and screening for ability to bind operator sequences.
  • the LHR of natural lac repressor protein can optionally be added to the C- terminus of the ligand binding protein to generate a protein that can form a looped DNA structure with increased repression.
  • the repressor proteins of the present invention can be produced from natural lac repressor protein by site-specific mutagenesis of the inducer binding region of the core domain and/or by random mutagenesis of the natural lac repressor protein.
  • the repressor proteins of the present invention can also be produced from repressor proteins with other ligand responsivity by replacement of their DNA recognition domain with that of lac repressor protein or alteration through mutagenesis of their DNA recognition domain to bind with high affinity to lac operator.
  • Figure 1 is a schematic of lactose operon.
  • the symbols correspond to the following: Pji promoter for i gene; i: gene encoding lactose repressor protein (Lad); p: promoter for lac enzymes; O: lactose operator sequence (LacO); z: gene encoding ⁇ - galactosidase; y: gene encoding lac permease; a: gene encoding thiogalactoside transacetylase; I: inducer; RNA pol: RNA polymerase.
  • Figure 2A is a schematic of a monomer of lac repressor protein.
  • Figure 2B is a schematic of the tetrameric structure of lac repressor protein, laid open to view the dimer-dimer assembly.
  • Figure 2C is a schematic of the tetrameric structure of lac repressor protein in its fully folded form.
  • Figure 2D is the x-ray crystallographic structure of the tetrameric lac repressor protein bound to operator (Lewis et al., 1996).
  • the present invention relates to substitutes for natural lac repressor protein.
  • the substitutes, or altered lac repressor proteins recognize and can bind to the lactose operator but have an altered ligand responsivity.
  • the altered ligand responsivity provides that a sugar or other small molecule other than allolactose or IPTG will, at reasonable concentrations, induce the altered lac repressor protein bound to operator DNA sites to undergo a conformational change such that affinity for the lac operator is diminished, and transcription from the adjacent promoter by RNA polymerase can take place.
  • An alternate embodiment of the invention provides an altered lac repressor with increased affinity for IPTG and wild-type operator DNA binding.
  • Sugars useful in the present invention are generally mono- and di-saccharides, such as arabinose, ribose, glucose, galactose and maltose. Preferred sugars are arabinose, ribose, D-glucose and D-galactose, and IPTG. Arabinose is a particularly preferred sugar.
  • Other small molecules useful in the present invention include amino acids, such as glutamine, leucine and ornithine; purines; pyrimidines; and small organic ions, among others.
  • Natural lac repressor protein is a homotetrameric protein of 150,000 Daltons with binding sites for four inducer molecules and two operator DNA sequences (Gilbert & Mueller-Hill, 1966; Riggs & Bourgeois, 1968; Butler et al., 1977; Whitson & Matthews, 1986). Each subunit is organized into two domains: a DNA-binding domain and a core domain (Lewis et al., 1996; Miller and Reznikoff, 1980). The DNA-binding domain has been identified with the N-terminal -60 amino acids and alone exhibits specificity, but low affinity, for operator DNA.
  • the DNA-binding domain contains a helix-turn-helix (HTH) motif homologous to other DNA binding proteins (Brennan & Matthews, 1989).
  • the core domain contains regions of the protein involved in the inducer binding site and in assembly of dimers and tetramers (Lewis et al., 1996; Friedman et al., 1995).
  • the region of the protein that forms the inducer binding site may hereinafter be referred to as the "ligand binding domain,” and the region of the protein involved in assembly of tetramers may hereinafter be referred to as the "tetramerizing domain.”
  • Figure 2 A shows a schematic of the lac repressor protein monomer
  • Figure 2B shows a simplified schematic of the tetramer structure generated by "opening" the tetramer to display the dimer-dimer configuration
  • Figure 2C shows a schematic of the fully folded configuration of the tetramer
  • Figure 2D is an x-ray crystallographic structure of the tetramer bound to operator (Lewis et al., 1996).
  • the tetramer structure of the lac repressor protein is a dimer-dimer assembly, the two dimers being aligned with their N-terminal domains on the same face of the molecule connected by a four-helical bundle at the base (Friedman et al., 1995; Lewis et al., 1996).
  • This four-helical bundle is the lac repressor protein dimer-dimer assembly motif and is localized to the C-terminal region from amino acids 342 to 356 (LHR).
  • Monomers of the lac repressor protein will not bind DNA (Daly & Matthews, 1986; Schmitz et al., 1976); therefore, at least a dimer is required to bind DNA.
  • S ⁇ Q ID NO:l gives the amino acid sequence of lac repressor protein (Beyreuther et al., 1975; Farabaugh, 1978), while S ⁇ Q ID NO:2 gives the amino acid sequence of arabinose binding protein (ABP) (Hogg & Hermodson, 1977).
  • the crystallographic structures of the periplasmic sugar binding proteins, such as ABP (Quiocho & Vyas, 1984), and the lac repressor protein indicate significant structural homology in their sugar binding sites (Lewis et al., 1996; Friedman et al., 1995; Nichols et al., 1995).
  • structural homology between two aligned sequences can be defined by minimum base change per codon (MBC/C) or amino acid homology per residue (AAH/R).
  • a random pair of sequences have an MBC/C of 1.45.
  • periplasmic sugar binding proteins and lac repressor have MBC/C of less than about 1.4, more preferably less than about 1.35, even more preferably less than about 1.3, and most preferably less than about 1.25.
  • a random pair of sequences also have an AAH/R of 4.46.
  • periplasmic sugar binding proteins and lac repressor have MBC/C of more than about 4.5, more preferably more than about 5.0, and most preferably more than about 5.5.
  • Altered lac repressor proteins can be made by combining the DNA-binding domain (HTH) and tetramerization domain (LHR) elements of lac repressor protein with a ligand binding protein, such as ABP. Such combining is accomplished by fusing the DNA encoding the HTH domain of the lac repressor protein to the DNA encoding the N- terminus of the ligand binding protein, ABP for example, with spacing chosen after visual inspection of aligned Lad and ABP structures. Recombinant DNA techniques are well known to those of skill in the art, and can be found, for example, in DNA Cloning: A Practical Approach (1995) or Molecular Cloning: A Laboratory Manual (1989). The DNA encoding the LHR domain from lac repressor protein is then added to sites carefully selected by visual inspection of the structure at the C-terminus of the ligand binding protein DNA.
  • HTH DNA-binding domain
  • LHR tetramerization domain
  • the LHR element can be left off for the construction of a dimeric repressor protein with altered responsivity to ligand.
  • the repressor protein in order to significantly bind the lactose operator, the repressor protein must form at least a dimer.
  • the resulting construct is a monomer. This monomeric construct can be subjected to multiple rounds of random mutagenesis in order to introduce the amino acid changes necessary for the new construct to form a dimer. The resulting mutants are then selected for ability to bind lactose operator.
  • Ligand binding proteins useful in the present invention include any protein which binds a sugar or other small molecule other than allolactose or IPTG and has substantial homology with the core domain, or ligand binding site, of natural lac repressor protein.
  • Examples of ligand binding proteins useful in the present invention are ABP, ribose binding protein (RBP) and D-glucose/D-galactose binding protein (GBP). Amino acid sequences for these binding proteins and their alignment with the lac repressor protein core domain can be found in the literature (Nichols et al., 1993). Similar ligand binding proteins with structural homology can be found by examining the family of periplasmic binding proteins from E coli.
  • ligand binding proteins can be found in, for example, Hsiao et al. (1996): glutamine-binding protein; Olah et al. (1993): leucine/isoleucine/valine-binding protein; Oh et al. (1993): lysine/arginine/ornithine- binding protein; Spurlino et al. (1991): maltose/maltodextrin-binding protein; Pflugrath & Quiocho (1988): sulfate-binding protein; Tarn & Saier (1993): various solute-binding receptors; and Matsuo & Nishikawa (1994): spermidine/putrescine-binding protein.
  • the ligand binding protein is ABP due to the ready availability and low cost of arabinose for use as an inducer.
  • Mutagenesis can be used to introduce amino acid changes that may result in oligomer formation.
  • chemical mutagenesis, or mutator strains to introduce nucleotide changes in vivo e.g., Stratagene, ⁇ picurian coli, XLl-Red
  • plasmid containing the altered lac repressor protein gene to be mutated is subjected to sequential rounds of mutagenesis and screening. After mutagenesis, the screening procedure delineated below is followed to test for an active repressor protein. Finally, the DNA from each of the positive colonies is sequenced.
  • Alternate screens known to those in the art e.g., using a toxic gene
  • the coding sequence for the altered lac repressor protein can be cloned into an expression vector.
  • the expression vector can then be placed into a bacterial or eukaryotic cell containing a reporter gene under lac operator control to test for an active repressor protein.
  • a reporter containing E. coli ⁇ - galactosidase encoded by lacZ
  • lacZ has been constructed by excising the lacZ gene under control of the lac operator from the plasmid pCR2.1 (Invitrogen) and inserting it into plasmid pACYCl 84 (New England Biolabs).
  • This new composite reporter plasmid is named pZCam and can be co-transformed or electroporated into bacteria with the plasmid containing the sequences of altered lac repressor proteins. Host cells for these plasmids should have no lactose repressor protein and no ⁇ -galactosidase (lacl ⁇ lacZ ' ). Next these bacteria are plated and treated with either methyl umbelliferyl- ⁇ ,D-galactoside (MUG) or 5-bromo-4-chloro-3-indolyl- ⁇ ,D-galactoside (X-gal) as indicators to determine whether an active repressor protein is present (white colonies).
  • MUG methyl umbelliferyl- ⁇ ,D-galactoside
  • X-gal 5-bromo-4-chloro-3-indolyl- ⁇ ,D-galactoside
  • colonies containing active repressor protein will be fluorescent (MUG) or blue (X-gal).
  • the coding sequence can be cloned into any expression vector known in the art and transformed into E coli that are lad ' , preferably Alacl, and lacpOz + .
  • the lacpOz region can be on a different, but compatible, plasmid or in the bacterial genome. Colonies containing active repressor protein will be white in the absence of an alternate inducer and fluorescent (MUG) or blue (X-gal) in the presence of an alternate inducer.
  • Specific amino acid changes that result in oligomer formation can be determined, and once a purified protein is obtained, the ability of the alternate sugar to diminish specific operator binding can be confirmed in vitro.
  • Purified proteins can be obtained by standard protein purification methods known to those of skill in the art.
  • altered lac repressor proteins can be made by site-specific and/or random mutagenesis of wild-type lac repressor.
  • Ligand binding proteins can be compared to the core domain, or inducer binding region, of the lac repressor protein. Differences in the two binding regions can be exploited to substitute residues characteristic of the alternate sugar binding protein into the lac repressor protein site. Selected amino acid changes can be introduced by site-specific mutagenesis.
  • Random mutagenesis methods can also be used to alter the ligand binding region of the lac repressor protein. Random mutagenesis of the lac repressor protein is followed by screens for altered ligand specificity and altered sensitivity to ligand in the protein products. Both site-specific and random mutagenesis methods are well known in the art and can be used in the practice of the present invention. Mutants can be screened by phenotypic analysis as described above. In the preferred forms, the plasmid containing the gene for altered lac repressor protein is either co-transformed with the reporter plasmid pZCam or is transformed into E coli that are
  • Alacl and / ⁇ cpOz + Introduction of wild-type lac repressor protein into bacteria containing pZCam or into the Alacl/lacpOz + bacteria results in white colonies in the presence of MUG or X-gal. In contrast, in the presence of IPTG, the colonies are fluorescent (MUG) or blue (X-gal).
  • Natural lac repressor protein subjected to mutation can be screened (1) for alternate inducer ligand response by substitution of an alternate inducer ligand or sugar for IPTG in this assay or (2) for sensitivity to ligand by using varied concentrations of IPTG or alternate inducer ligand.
  • Colonies that are white without ligand and become fluorescent (MUG)/blue (X-gal) in the presence of a specific alternate inducer ligand or IPTG at lower concentrations indicate that responsivity to ligand has changed.
  • MUG fluorescent
  • X-gal blue
  • other repressor proteins can be employed.
  • the DNA binding region of a repressor protein with alternate inducer ligand responsivity can be substituted with the DNA binding domain from lac repressor protein.
  • the N-terminal domain from the lac repressor protein is substituted for the DNA binding domain in a different repressor protein with alternate inducer ligand responsivity to generate a protein with ligand responsivity of the original protein but the capacity to recognize lac operator and regulate expression of genes under lac promoter-operator control.
  • the Lad family of regulatory proteins contains numerous potential targets (Weikert and Adhya, 1992), and others may be identified. Site-specific or random genetic alterations in the DNA binding domain of a repressor protein with alternate inducer ligand specificity can also be introduced to alter the recognition capacity to that for the lac operator sequence.
  • the altered lac repressor proteins of the present invention can be used in any manner natural lac repressor protein is used. Such uses include regulating expression of genes in bacterial cells and controlling expression and facilitating transcription in eukaryotic systems.
  • the altered lac repressor protein can be encoded on a plasmid and incorporated into a bacterial or eukaryotic cell.
  • altered lac repressor proteins can be used to control the expression of T7 polymerase, a protein that is unique to a specific phage that infects Escherichia coli.
  • T7 polymerase a protein that is unique to a specific phage that infects Escherichia coli.
  • a wide variety of proteins are expressed under control of the T7 polymerase promoter, e.g., p53 tumor suppressor protein, in cells where the T7 polymerase in turn is under lac repressor protein control.
  • the cost of turning on (inducing) the production of proteins under control of the lac repressor protein will be substantially reduced.
  • arabinose is the alternate inducer ligand
  • the cost of inducing the production of proteins under lac repressor protein control is diminished ⁇ 10-fold because of the differential between the cost of IPTG and arabinose.
  • a 10-fold increase in sensitivity to IPTG could decrease the cost of inducing protein production.
  • the HTH region of natural lac repressor protein can be fused to the N-terminus of ABP, and the LHR region of natural lac repressor protein fused to the C-terminus of ABP.
  • the coding sequence for the chimera can then be co-transformed with pZCam or electroporated into lad ' lacZ " E. coli.
  • the bacteria can be screened in the fluorescent/white (MUG) or blue/white (X-gal) screen described above to determine active repressor protein.
  • the HTH region of natural lac repressor protein can be fused to the N-terminus of ABP, without the LHR region at the C-terminus.
  • the resulting monomeric construct can be subjected to multiple rounds of random mutagenesis using PCR-based methods, chemical mutagenesis or mutator strains.
  • the coding sequence for the mutated chimera can then be co-transformed or electroporated into lad ' lacZ ' E. coli with pZCam.
  • the bacteria can be screened in the fluorescent/white (MUG) or blue/white (X-gal) screen described above to determine active repressor protein.
  • the DNA from each of the positive colonies can then be sequenced.
  • Natural lac repressor protein and ABP amino acid sequences can be compared to determine amino acid changes to be made. Natural lac repressor protein can then be subjected to site-directed mutagenesis to alter selected amino acids in the inducer binding region. Mutants of the lac repressor protein can be screened in the fluorescent/white
  • Natural lac repressor protein can be subjected to random mutagenesis, using XL-1 Red bacterial cells (Stratagene), chemical mutagenesis, or polymerase chain reaction methods, to alter the inducer binding region. Mutants of the lac repressor protein can then be screened in the fluorescent/white (MUG) or blue/white (X-gal) assay as described above to detect responsivity to various sugars and other small molecules.
  • MMG fluorescent/white
  • X-gal blue/white
  • the altered lac repressor proteins with responsivity to arabinose produced as in Examples 1 - 4 can be used to control the expression of lac repressor protein-regulated T7 polymerase in E. coli, as found in multiple commercial vectors, by introducing the plasmid encoding the altered lactose repressor protein on a plasmid compatible with the other constructs present.
  • Arabinose or alternate ligand presence or decreased concentrations of IPTG compared to that required by wild type protein result in decreased affinity for the lac operator and consequent production of mRNA for any gene under control of the T7 recognition sequence.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention a trait à des protéines modifiées répresseur de lac reconnaissant le gène opérateur du lactose mais dont la sensibilité du ligand est modifiée. Cette modification de sensibilité fait qu'un sucre ou une autre petite molécule autre qu'une allolactose ou une isopropyl-β,D-thiogalactoside (IPTG) agit en tant qu'inducteur de la protéine modifiée répresseur de lac ou que cette IPTG agit à des concentrations moins importantes. Elle porte également sur des séquences d'ADN codant les protéines modifiées répresseur de lac ainsi que sur des cellules bactériennes et des cellules eucaryotes renfermant les protéines modifiées répresseur de lac.
PCT/US1998/024949 1997-11-20 1998-11-20 Proteine represseur du lactose a sensibilite du ligand modifiee WO1999027108A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU14673/99A AU1467399A (en) 1997-11-20 1998-11-20 Lactose repressor proteins with altered ligand responsivity
US10/197,053 US7390645B2 (en) 1997-11-20 2002-07-17 Lactose repressor proteins with increased operator DNA binding affinity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6621397P 1997-11-20 1997-11-20
US60/066,213 1997-11-20

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US09554537 A-371-Of-International 2000-05-12
US73683600A Continuation-In-Part 1997-11-20 2000-12-14
US10/197,053 Continuation-In-Part US7390645B2 (en) 1997-11-20 2002-07-17 Lactose repressor proteins with increased operator DNA binding affinity

Publications (1)

Publication Number Publication Date
WO1999027108A1 true WO1999027108A1 (fr) 1999-06-03

Family

ID=22068020

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/024949 WO1999027108A1 (fr) 1997-11-20 1998-11-20 Proteine represseur du lactose a sensibilite du ligand modifiee

Country Status (2)

Country Link
AU (1) AU1467399A (fr)
WO (1) WO1999027108A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8257956B2 (en) 2008-10-28 2012-09-04 E. I. Du Pont De Nemours And Company Sulfonylurea-responsive repressor proteins
US8288127B2 (en) 2003-11-19 2012-10-16 Pfenex, Inc Protein expression systems
KR101527349B1 (ko) * 2013-01-25 2015-06-17 전남대학교산학협력단 유도체에 비의존적으로 개량된 전사인자 AraC 및 이의 용도

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT397812B (de) * 1992-12-18 1994-07-25 Polymun Scient Gmbh Verfahren zur expression von genen unter kontrolle des lac-operators
EP0685560A2 (fr) * 1994-06-01 1995-12-06 Suntory Limited Méthode pour contrôler l'expression génétique
WO1997004110A1 (fr) * 1995-07-14 1997-02-06 Somatogen, Inc. Procedes d'accroissement de l'expression de proteines
WO1997049813A2 (fr) * 1996-06-26 1997-12-31 Cambia Biosystems Llc Represseurs de glucoronides et leur utilisation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT397812B (de) * 1992-12-18 1994-07-25 Polymun Scient Gmbh Verfahren zur expression von genen unter kontrolle des lac-operators
EP0685560A2 (fr) * 1994-06-01 1995-12-06 Suntory Limited Méthode pour contrôler l'expression génétique
WO1997004110A1 (fr) * 1995-07-14 1997-02-06 Somatogen, Inc. Procedes d'accroissement de l'expression de proteines
WO1997049813A2 (fr) * 1996-06-26 1997-12-31 Cambia Biosystems Llc Represseurs de glucoronides et leur utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FIECK ET AL: "Modifications of the E. coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear acumulation", NUCLEIC ACIDS RESEARCH, vol. 20, no. 7, 1992, pages 1785 - 1791, XP002095852 *
NICHOLS ET AL: "Model of Lactose Repressor Core Based on Alignment with Sugar-binding Proteins Is Concordant with Genetic and Chemical Data", JOURNAL OF BIOLOGICAL CHEMSITRY, vol. 268, no. 23, 15 August 1993 (1993-08-15), pages 17602 - 17612, XP002095853 *
WEICKERT AND ADHYA: "A Family of Bacterial Regulators Homologous to Gal and Lac Repressors", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 22, 5 August 1992 (1992-08-05), pages 15869 - 15874, XP002096157 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8288127B2 (en) 2003-11-19 2012-10-16 Pfenex, Inc Protein expression systems
US8257956B2 (en) 2008-10-28 2012-09-04 E. I. Du Pont De Nemours And Company Sulfonylurea-responsive repressor proteins
US8580556B2 (en) 2008-10-28 2013-11-12 E. I. Du Pont De Nemours And Company Sulfonylurea-responsive repressor proteins
US8877503B2 (en) 2008-10-28 2014-11-04 E. I. Du Pont De Nemours And Company Sulfonylurea-responsive repressor proteins
KR101527349B1 (ko) * 2013-01-25 2015-06-17 전남대학교산학협력단 유도체에 비의존적으로 개량된 전사인자 AraC 및 이의 용도

Also Published As

Publication number Publication date
AU1467399A (en) 1999-06-15

Similar Documents

Publication Publication Date Title
Jamieson et al. In vitro selection of zinc fingers with altered DNA-binding specificity
Koudelka et al. Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro
US7625700B2 (en) In vivo library-versus-library selection of optimized protein-protein interactions
Consler et al. Role of proline residues in the structure and function of a membrane transport protein
JP4493271B2 (ja) Ｃ型レクチン様ドメインの骨格構造を有する蛋白質のコンビナトリアルライブラリ
Clavel et al. Expression of the tolQRA genes of Escherichia coli K‐12 is controlled by the RcsC sensor protein involved in capsule synthesis
Kolmar et al. Membrane insertion of the bacterial signal transduction protein ToxR and requirements of transcription activation studied by modular replacement of different protein substructures.
US20080108789A1 (en) DNA & protein binding miniature proteins
KR20080035603A (ko) 융합 폴리펩티드의 공번역 전좌를 사용한 파아지디스플레이
EP1727904B1 (fr) Methode a base de peptides pour suivre l'expression genetique dans une cellule hote
Webber et al. Involvement of the amino‐terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli
JP3723240B2 (ja) 遺伝子発現の制御方法
WO1999027108A1 (fr) Proteine represseur du lactose a sensibilite du ligand modifiee
US7390645B2 (en) Lactose repressor proteins with increased operator DNA binding affinity
TW201309720A (zh) 胜肽庫
Hummel et al. A functional protein hybrid between the glucose transporter and the N‐acetylglucosamine transporter of Escherichia coli
Groch et al. Determination of DNA‐binding parameters for the Bacillus subtilis histone‐like HBsu protein through introduction of fluorophores by site‐directed mutagenesis of a synthetic gene
Oertel-Buchheit et al. Isolation and characterization of LexA mutant repressers with enhanced DNA binding affinity
Szeto et al. A conserved polar region in the cell division site determinant MinD is required for responding to MinE-induced oscillation but not for localization within coiled arrays
Oertel-Buchheit et al. Genetic analysis of the LexA repressor: isolation and characterization of LexA (Def) mutant proteins
EP1198586B1 (fr) Selection in vivo bibliotheque contre bibliotheque d'interactions proteine-proteine optimisees
US7235385B2 (en) Methods for enhancing expression of recombinant proteins
Nam et al. DNA light-strand preferential recognition of human mitochondria transcription termination factor mTERF
Eng et al. The C-terminus of MinE from Neisseria gonorrhoeae acts as a topological specificity factor by modulating MinD activity in bacterial cell division
BINDING I IIIII 1111111111111111111111111111111111111111111111111111111111111

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG 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 BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 09554537

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA