WO1999027108A1

WO1999027108A1 - Lactose repressor proteins with altered ligand responsivity

Info

Publication number: WO1999027108A1
Application number: PCT/US1998/024949
Authority: WO
Inventors: Kathleen S. Matthews; Liskin Swint-Kruse
Original assignee: William Marsh Rice University
Priority date: 1997-11-20
Filing date: 1998-11-20
Publication date: 1999-06-03
Also published as: AU1467399A

Abstract

The present invention provides altered lac repressor proteins that recognize 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 acts as an inducer for the altered lac repressor protein or that IPTG acts at lower concentrations. DNA sequences encoding the altered lac repressor proteins and bacterial and eukaryotic cells containing altered lac repressor proteins are also provided.

Description

LACTOSE REPRESSOR PROTEINS WITH ALTERED LIGAND

RESPONSIVITY

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,

OO ), which binds with high affinity to the partially two-fold symmetric lac operator

DNA target sequence (O) that overlaps the promoter sequence (p). RNA polymerase binding, initiation, and/or elongation are inhibited when Lad occupies this site, precluding production of the mRNA encoding the lac enzymes (z, y, a). In the absence of lactose, the high affinity of Lad for LacO allows production of only small quantities of lacZYA mRNA. In the presence of inducer sugars (I), a conformational change in Lad (depicted as (O-»D) (Lewis et al., 1996) reduces operator binding affinity without effect on binding to nonspecific DNA (Lin & Riggs, 1975). Excess non-operator DNA in the cell thereby effectively competes for binding to the protein and sequesters the repressor-inducer complex, allowing transcription of downstream lac mRNA to proceed as long as inducer sugar is available. When inducer sugar levels are depleted, inducer dissociates from the repressor protein, which resumes its conformation with high affinity for operator DNA, associates with LacO, and shuts down further synthesis of lac mRNA. The lactose regulatory cycle therefore involves association with both specific and non-specific DNA sequences, binding of sugar molecules, and conformational shifts in response to these ligands. 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 β- galactosidase result in production of the in vivo inducer, β-l,6-allolactose. This sugar is the natural inducer of Lad (Jobe & Bourgeois, 1972). When lactose levels are decreased, the intracellular store of natural inducer is depleted by β-galactosidase hydrolysis. These enzymatic activities ensure that the lac enzymes are not expressed except in the presence of lactose. A different inducer, isopropyl-β,D-thiogalactoside (IPTG), that is not a substrate for β-galactosidase, is generally used to turn on transcription of genes cloned under control of the lac promoter-operator (in place of the z, y, and a genes depicted in Figure 1). However, IPTG is an expensive sugar, causing systems employing Lad as a control protein for expression to be costly. Different, unique repressor proteins that recognize lac operator sequence, yet respond to binding of an alternate, less expensive inducer ligand, would significantly reduce the cost of gene expression. Similarly, an altered repressor protein with increased affinity for IPTG would diminish these costs. Such repressor proteins can be applied to any system in which lac operator is used as the target sequence for transcriptional control and will provide significant cost savings in the generation of cloned protein products.

The following definitions are provided in order to aid those skilled in the art to understand the detailed description of the present invention.

"ABP" refers to arabinose binding protein. "HTH" refers to the helix-turn-helix domain of lac repressor protein.

"IPTG" refers to isopropyl-β,D-thiogalactoside.

"LHR" refers to the leucine heptad repeat domain of lac repressor protein.

"MUG" refers to methyl umbelliferyl-β,D-galactoside. j -

"X-gal" refers to 5-bromo-4-chloro-3-indolyl-β,D-galactoside.

The phrase "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. The phrase "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. The phrase "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.

The phrase "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.

The phrase "substantial 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. In a preferred embodiment, 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. Alternatively, 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.

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

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 β- 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, and 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, while 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.

Homology has been noted between monomeric periplasmic sugar binding proteins in E coli and the core domains of repressor proteins (Mueller-Hill, 1983; Sams et al., 1984; Nichols et al., 1993). SΕQ ID NO:l gives the amino acid sequence of lac repressor protein (Beyreuther et al., 1975; Farabaugh, 1978), while SΕQ 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). As known to one skilled in the art and disclosed by 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. Preferably, 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. Preferably, 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. The structures reported by Lewis et al., 1996; Friedman et al., 1995; and Nichols et al., 1995, show that the ligand binding regions consist of two subdomains that surround a binding site. Multiple residues throughout the protein sequences participate in ligand contacts within this site. The homology between monomeric periplasmic sugar binding proteins and the inducer binding region of the core domain of the lac repressor protein allows alteration of natural lac repressor protein such that the repressor protein will maintain its former native DNA binding affinity, but have an altered ligand responsivity by binding to a new inducer other than allolactose or IPTG or by binding with higher affinity to IPTG.

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.

The LHR element can be left off for the construction of a dimeric repressor protein with altered responsivity to ligand. However, as described previously, in order to significantly bind the lactose operator, the repressor protein must form at least a dimer. When monomeric ligand binding proteins are fused to the DNA-binding domain of the lac repressor protein, 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. Various 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.

Preferably, 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. Using PCR-based methods, chemical mutagenesis, or mutator strains to introduce nucleotide changes in vivo (e.g., Stratagene, Εpicurian 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) can also be used which will result in determining active repressor proteins.

To test an altered lac repressor protein, 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. For example, a reporter containing E. coli β- galactosidase (encoded by 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 β-galactosidase (lacl^~ lacZ^'). Next these bacteria are plated and treated with either methyl umbelliferyl-β,D-galactoside (MUG) or 5-bromo-4-chloro-3-indolyl-β,D-galactoside (X-gal) as indicators to determine whether an active repressor protein is present (white colonies). In the presence of the inducer of interest (i.e., arabinose, if ABP is the ligand binding protein), colonies containing active repressor protein will be fluorescent (MUG) or blue (X-gal). Alternatively, 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.

Alternatively, 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 /αcpOz⁺. 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. As many ligands or concentrations can be screened as desired. In addition, other repressor proteins can be employed. Alternatively, 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. For example, 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. 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.

By using altered lac repressor proteins, the cost of turning on (inducing) the production of proteins under control of the lac repressor protein will be substantially reduced. For example, when 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. Similarly, a 10-fold increase in sensitivity to IPTG could decrease the cost of inducing protein production.

The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art will appreciate that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1

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. Following mutagenesis using PCR-based methods, chemical mutagenesis or mutator strains to produce oligomer formation, the bacteria can be screened in the fluorescent/white (MUG) or blue/white (X-gal) screen described above to determine active repressor protein. Example 2

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.

Example 3

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

(MUG) or blue/white (X-gal) assay as described above to detect responsivity to arabinose. Example 4

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. Example 5

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.

All of the 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.

REFERENCES The following references (and references contained therein), to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

CLAIMS:

I. A repressor protein of the lac operator comprising a DNA-binding domain of natural lac repressor protein and a ligand binding domain with responsivity to an alternate inducer ligand or altered responsivity to IPTG. 2. The repressor protein of claim 1 wherein the alternate inducer ligand is a sugar.

3. The repressor protein of claim 1 wherein the alternate inducer ligand is arabinose.

4. The repressor protein of claim 1 wherein the alternate inducer ligand is ribose.

5. The repressor protein of claim 1 wherein the alternate inducer ligand is D-glucose or D-galactose. 6. The repressor protein of claim 1 with increased affinity for IPTG.

7. The repressor protein of claim 1 further comprising a tetramerizing domain of natural lac repressor protein.

8. The repressor protein of claim 7 wherein the alternate inducer ligand is a sugar.

9. The repressor protein of claim 7 wherein the alternate inducer ligand is arabinose. 10. The repressor protein of claim 7 wherein the alternate inducer ligand is ribose.

I I . The repressor protein of claim 7 wherein the alternate inducer ligand is D-glucose or D-galactose.

12. The repressor protein of claim 7 with increased affinity for ligand IPTG.

13. A DNA sequence encoding the repressor protein of any one of claims 1 - 12. 14. A bacterial or eukaryotic cell containing the repressor protein of any one of claims 1 - 12 or DNA encoding the repressor protein of any one of claims 1 - 12.

15. The bacterial cell of claim 14 which is an E coli cell.

16. A method for preparing an altered lac repressor protein comprising fusing a DNA- binding domain of natural lac repressor protein to the N-terminus of a ligand binding protein having responsivity to a ligand other than allolactose or IPTG.

17. The method of claim 16 further comprising fusing a tetramerizing domain of natural lac repressor protein to the C-terminus of the ligand binding protein.

18. The method of claim 16 wherein the ligand binding protein is arabinose binding protein.

19. A method for preparing an altered lac repressor protein comprising altering a non- lac repressor protein having structural homology to natural lac repressor protein and altered inducer ligand responsivity.

20. The method of claim 19 wherein the altering is performed by substituting a DNA- binding domain of the natural lac repressor protein for a corresponding domain of the non- lac repressor protein.