Abstract:
An expression system for transforming  E. coli  with a nucleic acid molecule of interest has an operator sequence of a cmt operon operatively linked to a promoter for the operator, and, a repressor sequence from a cym operon operatively linked to a promoter for the repressor. The expression system may have a nucleic acid molecule of interest, for example, a nucleic acid molecule that encodes a protein. Any type of  E. coli  host cells may be transformed with the expression system. A method of producing a protein involves transforming an  E. coli  host cell with the expression system having a nucleic acid molecule that codes for a protein, and, culturing the host cell in a culture medium under conditions in which the nucleic acid molecule will express the protein.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/929,389 filed Jun. 25, 2007, the entire contents of which is herein incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to biotechnology, in particular to gene expression systems in  Escherichia coli.    
       BACKGROUND OF THE INVENTION 
       [0003]    New hosts and expression vectors for the production of industrially important recombinant protein are continuously being developed for the purpose of increasing production yields and simplifying down stream processes such as single-step purification using affinity tag systems. Though many expression hosts are available,  Escherichia coli  continues to remain one of the most frequently employed host for the mass production of various useful recombinant proteins or peptides, and many promoters such as P lac , P trp , P tac , λP L , P T7  and P BAD  are commonly utilized for the construction of expression vectors (Baneyx, 1999). Among these, lacUV5, tac and combined system of P T7  with lacUV5 are widely used, because the expression can easily be regulated by varying the concentration of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG, Schein and Noteborn, 1988). However, the use of IPTG precludes the use of these expression systems in pilot scale production of recombinant proteins, mainly due to the high cost and potential toxicity of IPTG (Figge et al., 1988, Kosinski et al., 1992, Bhandari and Gowrishankar, 1997, Leigh et al., 1998, Yogender et al., 2001, Wang et al., 2004). Other promoters called λPL and λPR are generally induced by a temperature shift, which can have an adverse effect on the protein folding and reduce the final yield of the product (Remaut et al., 1981). 
         [0004]    It is known that the expression of a homologous or heterologous gene may be enhanced by replacing a promoter sequence naturally associated with that gene with a strong promoter sequence, which results in an enhanced expression of the gene at the transcriptional level (Studier and Moffatt, 1986, Gupta et al., 1999). However, ideal expression system should provide high-level expression under induced conditions and no basal expression under repressed conditions, yet should show adjustability to intermediate levels over a wide range of inducer concentrations (Rossi and Blau, 1998, Keyes and Mills, 2003). To date, only a limited number of expression system have been explored for the industrial recombinant protein production. The field of modern biotechnology is competitive and is attracting considerable interest from industrial partners outside the traditional fermentation industry, interested in the industrial applications of enzymes and other proteins. Therefore, it is not surprising that several of these partners have started to explore the possibility of using new expression systems as alternatives to those covered by patents and patent application (Staub, et al., 2002). It is in the interest of the biotechnological industry to seek new expression systems, which are easily accessible, cheap and simple to regulate. Especially, systems that are independent of the host strain, medium, and growth rate are needed. Therefore, the aim of our work was to develop a next generation of a novel expression system which fulfills most of factors to be an ideal expression system of  E. coli.    
         [0005]    The ability to produce high biomass densities of  E. coli  in fermentors (Lee, 1996, Thiry and Cingolani, 2002), combined with the newly adopted regulatory genetic elements obtained from  Pseudomonas putida  F1 (Choi et al., 2006), renders this novel expression system extremely interesting as a potential tool for the production of recombinant proteins and of industrially important bulk chemicals. The applications of such an expression system is equally comprehensive encompassing the: (1) production of research reagents to support R&amp;D in biotechnology and in various biological fields including proteomics; (2) production of commercial recombinant proteins (enzymes and bio-active peptides); (3) production of various biomaterials including proteinaceous and non-proteinaceous bio intermediates; (4) as a tool for metabolic engineering work. 
         [0006]    International Patent Publication WO 2007/022623 published Mar. 1, 2007 discloses the use of regulating elements from  Pseudomonas putida  to enable inducible regulation of gene expression in  Methylobacterium extorquens . International Patent Publication WO 2006/037215 published Apr. 13, 2006 discloses the use of cumate inducible regulating elements to enable inducible regulation of gene expression in Chinese Hamster Ovary (CHO) cells. In both of these cases, the repressor and its weak promoter are incorporated into the genome of the host cell separately from the plasmid containing the gene of interest, operator and promoter for the operator. 
         [0007]    There is a need for a tightly regulated, inducible gene expression system in  Escherichia coli.    
       SUMMARY OF THE INVENTION 
       [0008]    A novel inducible expression system, designated pNEW, is disclosed carrying a synthetic operator of  Pseudomonas putida  and expression profiles of nucleic acid molecules of interest. The expression system comprises an operator and repressor complex that is activated by cumate and like inducers, leading to regulated gene expression over several orders of magnitude. 
         [0009]    Thus, there is provided an expression system for transforming  E. coli  with a nucleic acid molecule of interest, the vector comprising: an operator sequence of a cmt operon operatively linked to a promoter for the operator; and, a repressor sequence from a cym operon operatively linked to a promoter for the repressor. 
         [0010]    The expression system may further comprise the nucleic acid molecule of interest, which may be, for example, an antisense inhibitor of gene expression, a nucleic acid coding for a protein, or any other nucleic acid molecule for which expression is desired in  E. coli . Preferably, the nucleic acid molecule encodes a protein. 
         [0011]    There is further provided an  E. coli  host cell transformed with an expression system of the present invention. 
         [0012]    There is further provided a method of producing a protein comprising transforming an  E. coli  host cell with an expression system of the present, the nucleic acid molecule of the expression system coding for a protein; and, culturing the host cell in a culture medium under conditions in which the nucleic acid molecule will express the protein. 
         [0013]    Expression of the nucleic acid molecule of interest in  E. coli  is activated by addition of an inducer. The inducer may comprise, for example, p-cumate, butyrate, dimethyl-p-aminobenzoic acid (DM PABA), trimethyl cumate, ethylbenzoate, a salt thereof or a combination thereof. p-Cumate is preferred. 
         [0014]    A tightly regulated gene expression system in  Escherichia coli  of the present invention may include regulatory elements of the  Pseudomonas putida  F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-cumate as an inducer in any type of  E. coli  strains. This expression system includes a specific expression vector, pNEW, that may contain a partial T5 phage promoter combined with the P1 synthetic operator and the cymR repressor protein encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer, e.g. p-cumate, which is non-toxic, inexpensive and easy to use. High concentrations of recombinant protein accumulation are observed (generally, 40-85% of total cellular protein), which is a more than 10,000-fold induction in stably transformed cells on average. Both high induction of transcription and extremely low basal expression allowed extremely high induction levels, with a degree of control that is far superior to other currently available  E. coli  expression systems, for example the T7 system with IPTG inducer. The results indicated that the present pNEW expression system is a highly efficient system for the potential production of recombinant proteins in any type of  E. coli  strains, especially when cloned proteins have growth inhibitory or toxic effects to host cell metabolism. 
         [0015]    Further features of the invention will be described or will become apparent in the course of the following detailed description. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]    In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which: 
           [0017]      FIG. 1  is a schematic diagram of the mechanism of action of the cumate-switchable expression system; 
           [0018]      FIG. 2  is a physical map of plasmid pNEW-gfp designed for regulated expression of heterologous gene in  E. coli;    
           [0019]      FIGS. 3A and 3B  depict culture plate assays (A) and liquid culture assays (B) showing regulated expression of GFP in various  E. coli  strains as lost; 
           [0020]      FIG. 4  depicts a comparison between T7 system and cumate system for green fluorescent protein (GFP) expression in plates containing IPTG (1 mM) and cumate (0.12 mM) as inducer, respectively; 
           [0021]      FIG. 5  is a physical map of pNEW-PhaC1, 2 and microscopic observation of the recombinant strains upon cumate induction (0.12 mM); 
           [0022]      FIG. 6  depicts culture plates showing heterologous gene expression of esterase in  E. coli  Top10 using cumate expression system of the present invention without and with cumate as inducer; 
           [0023]      FIG. 7  depicts an expression profile of recombinant β-galactosidase on SDS-PAGE; 
           [0024]      FIG. 8  depicts SDS-PAGE profile of soluble and insoluble fractions in the production of synthetic thrombin inhibitor peptide using carrier protein (SFC120) to form fusion peptide in  E. coli;    
           [0025]      FIGS. 9A-9F  depict fermenters at various stages of cell culture for recombinant  E. coli  cultures induced with IPTG or cumate; and, 
           [0026]      FIGS. 10A and 10B  are graphs depicting time course green fluorescent protein (GFP) yield comparisons between T7 system and cumate system at concentrations of 100 μm inducer (A) and 1000 μm inducer (B). 
       
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Materials and Methods 
       [0027]    Bacterial strains and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1.  E. coli  strains DH5α, S17-1 λ/pir, K12, Top10, and BL21(DE3), were used for the heterologous gene expression host. Especially,  E. coli  strain Top10 was used for cloning and propagation of recombinant DNA and some target protein expression host.  E. coli  was cultured in Luria Bertani broth (LB) at 37° C. and media were solidified by 1.8% agar (Difco) when appropriate. Antibiotics were used at the following concentrations (in μg/ml): ampicillin, 100; kanamycin (Km), 50; tetracyclin (Tc), 35. 
         [0028]    Benchtop Fermentations. Batch fermentation experiments were carried out in a 14-I bioreactor (BioFlo 110, New Brunswick Scientific, Edison, N.J. USA) to compare GFP production yield between T7 expression system and cumate system. For the batch culture, pre-cultures were used to inoculate the bioreactor filled with 5 l of medium A (Yoon et al, 2003) and initial O.D. was adjusted to 0.1 for both expression systems. The cultures induced with IPTG for T7 system and cumate for cumate system when O.D. reached at 38 to 42. For cultures carried out in bioreactors, pH and dissolved oxygen were controlled at 7 and 25%, respectively. 
         [0029]    Construction of expression vector. The operator sequence of cmt operon from  P. putida  F1 was introduced downstream of the phage T5 promoter (Bujard, et al. 1987) by polymerase chain reaction (PCR). The pNEW regulative expression vector was obtained in several steps: first, the P T5  synthetic operator sequence (OP)-GFP was PCR-amplified from pCUM-gfp (Choi, et al. 2006) using primers 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    AAA CAG ACA ATC TGG TCT GTT TGT ATT AT-3′) (SEQ ID NO: 1) (the SacI site is underlined, partial T5 promoter is boxed and operator site is in bold) and GFP-SPH-R (5′-C GC ATG C TC AGT TGT ACA GTT CAT CCA TGC C-3′) (SEC) ID NO: 2) (the SphI site is underlined). The 954 by PCR fragment containing P T5 -operator-gfp was cloned into pCR2.1 to create pCR-T5OP. Next, a 954 by SacI-SphI fragment from pCR-T5OP was then ligated between the SacI-SphI sites of pET36 (Novagen) to form pNEW-pre. 
         [0030]    Subsequently, the P km -cymR was amplified by PCR from pBRI-cymR1 (Choi et al. 2006) using primers PKM-CYM-MLU-F (5′-C AC GCG T CC GGA ATT GCC AGC TGG GGC GCC CTC TGG TAA GGT TGG GAA GCC CTG CAA AGT AAA CTG GAT GGC TTT CTT GCC GCC AAG GAT CTG ATG GCG CAG GGG ATC AAG ATC TGA TCA AGA GAC AGG ATG AGG ATC GTT TCG CAA GAT GGT GAT CAT GAG TCC AAA GAG AAG AAC ACA G-3′) (SEQ ID NO: 3) (the MluI site is underlined) and CYM-PCI-R (5′-C AC ATG T CT AGC GCT TGA ATT TCG CGT ACC GCT CTC-3′) (SEQ ID NO: 4) (the PciI site is underlined). The PCR product containing P km -cymR was then cloned into pCR2.1 to create pCR-Pkm-cymR, and MluI-PciI fragment from pCR-P km -cymR was ligated to the pNEW-pre digested by the same enzymes to generate pNEW-gfp. 
         [0031]    Other reporter gene cloning. In order to validate heterologous protein production using newly developed cumate switch system (pNEW system), we have tested GFP, polyhydroxyalkanoic acids synthetase (PhaC1 and PhaC2), lactase, esterase and synthetic thrombin inhibitory peptides. To clone PhaC1 and PhaC2 genes from  Pseudomonas fluorescens  GK13, the genomic DNA was isolated, and the chromosome was subjected to PCR using the primers PhaC1FNhe (5′-C GC TAG C AT GAG CAA CAA GAA CAA TGA AGA CCT GCA GCG C-3′) (SEQ ID NO: 5) (the NheI site is underlined), PhaC1RMFE (5′-G CA ATT G TC AAC GTT CGT GGA CAT AGG TCC CTG G-3′) (SEQ ID NO: 6) (the MfeI site is underlined), for PhaC1 and PhaC2FNhe (5′-C GC TAG C AT GCG AGA GM ACA GGT GTC GGG AGC CTT G-3′) (SEQ ID NO: 7) (the NheI site is underlined), PhaC2RCla (5′-G CA ATT G TC AGC GCA CGT GCA CGT AGG TGC CGG G-3′) (SEQ ID NO: 8) (the ClaI site is underlined) for PhaC2 to obtain 1680-bp and 1683-bp PCR products, respectively. The PCR products were digested with NheI and MfeI (PhaC1) and with NheI and ClaI (PhaC2), and cloned into pNEW-gfp digested with same restriction enzymes to generate pNEW-phaC1 and pNEW-phaC2, respectively. The 2,100 by fragment carrying the lactase gene (bgl) from  Bifidobacterium infantis  was amplified from pEBIG4 (Hung et al. 2001) using primers BGL-F-Nhe (5′-C GC TAG C AT GGA ACA TAG AGC GTT CAA GTG G-3′) (SEQ ID NO: 9) (the NheI site is underlined) and BGL-R-Sac (5′-C GA GCT C TT ACA GCT TGA CGA CGA GTA CGC CG-3′) (SEQ ID NO: 10) (the SacI site is underlined). For the amplification of esterase gene (1,800 bp, estI) from  Lactobacillus casei , pCESTa (Choi, et al. 2004) was used as a template with primers EST-F-Nhe (5′-C GC TAG C AT GGA TCA ATC TAA AAC AAA TCA AAA C-3′) (SEQ ID NO: 11) (the NheI site is underlined) and EST-R-Sac (5′-C GA GCT C TT ATT TAT TTG TAA TAC CGT CTG C-3′) (SEQ ID NO: 12) (the SacI site is underlined). These NheI-SacI fragments of bgl and est were then replaced with a gfp gene in the pNEW-gfp to form pNEW-bgl and pNEW-est, respectively. To amplify synthetic thrombin inhibitor peptide encoding gene with carrier protein (SFC120), pTSN-6A (Osborne et al., 2003) was used as a template with primers MFH-FNhe (5′-C GC TAG C AT GGC AAC TTC AAC TAA AAA ATT AC-3′) (SEQ ID NO: 13) (the NheI site is underlined) and MFH-RMfe (5′-G CA ATT G TT ATT GTA AAT ACT CTT CTG GAA TCG G-3′) (SEQ ID NO: 14) (the MfeI site is underlined). The PCR product was digested with NheI and MfeI and the 456 by fragment encoding carrier protein with synthetic thrombin inhibitor peptide was cloned into pNEW-gfp digested with same restriction enzymes to generate pNEW-mfh. 
         [0032]    Host cell transformation and gene expression. pNEW vectors harbouring different genes of interest were transformed into various  E. coli  cells by chemical or electroporation methods (Sambrook and Russell, 2000). The transformed cells were grown at 37° C. in LB medium, and expression of genes under developed system was induced with 20 μg/ml cumate or as indicated. 
         [0033]    Detection of gene expression. Detection of GFP was carried out by fluorescence microscopy, and quantified by using a SPECTRAFluor Plus (TECAN Austria Gmbh, Grodïg, Austria) under excitation and emission wavelengths of 485 and 508 nm, respectively. Concentration of GFP was calculated based on a linear relationship between concentration and fluorescence units determined using solutions of purified GFP (Qbiogene). The biomass (X) was determined by cell dry weight measurement of the samples (Moisture Analyzer MA 30, Sartorius). 
         [0034]    Esterase activity was determined by a spectrophotometric method using paranitrophenyl caprylate (pNP-caprylate) as substrate. The rate of hydrolysis of pNP-caprylate at 37° C. was measured in 50 mM sodium phosphate buffer (pH 7.0) according to the method described previously (Kademi et al., 1999). One unit of activity was defined as the amount of enzyme that liberated 1 μmol of p-nitrophenol per min under the given assay conditions. The β-galactosidase activity was measured with o-nitrophenol-β-D-galactoside (ONPG) as a substrate and one unit of activity was defined as the amount of enzyme that liberated 1 μmol of o-nitrophenol per min (Sambrook and Russel, 2000). The protein concentration was estimated by the method of Bradford (Bradford, 1976) using the Bio-Rad protein assay kit with bovine serum albumin as a standard. 
         [0035]    Western blotting. Integrative expression of repressor protein (cymR) was determined by western blotting using standard protocol. cymR was detected with rabbit anti-bCymR #422 antibody (0.1 g ml −1 ) and a goat anti-rabbit IgG (H+L) HRP conjugate (0.1 μg ml −1 ; Pierce cat#31460, West Grove, Pa.). Cells were lysed in SDS-PAGE sample buffer. 
         [0000]    
       
         
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Strains and Plasmids 
               
             
          
           
               
                 Strain or plasmid 
                 Description 
                 Reference or Source 
               
               
                   
               
             
          
           
               
                   Pseudomonas  strains 
               
             
          
           
               
                 fluorescens GK13 
                 Source of PhaC1 and C2 genes 
                 Jaeger, et al., 1995 
               
               
                 putida F1 
                 Origin of cymR gene and operator 
                 Eaton, 1997 
               
               
                   
                 sequence in the cmt operon, 
               
               
                   
                 respectively. 
               
             
          
           
               
                   E. coli  strains 
               
             
          
           
               
                 S-17Iλ pir 
                 Tp r  Sm r , recA thi pro hsdR M +  RP4: 2- 
                 De Lorenzo et al., 1993 
               
               
                   
                 Tc:Mu:Km Tn7 λpir 
               
               
                 Top10 
                 F− mcrA Δ(mrr-hsdRMS-mcrBC) 
                 Grant et al., 1990 
               
               
                   
                 φ80lacZΔM15 ΔlacX74 recA1 
               
               
                   
                 araD139 Δ(araleu) 
               
               
                   
                 7697 ga/U ga/K rpsL (StrR) endA1 
               
               
                   
                 nupG 
               
               
                 BL21(DE3)PLyS 
                 F −  ompT gal dcm Ion hsdS B (r B   −  m B   − ) 
                 Novagen 
               
               
                   
                 λ(DE3) pLysS(cm R ) 
               
               
                 DH5α 
                 endA1 recA1 hsdR17(r K   −  m K   + ) 
                 Hanahan, 1985 
               
               
                   
                 supE44 thi-1 gyrA96 φ80dlaCZΔM15 
               
               
                   
                 Δ(lacZYA-argF)U169 λ −   
               
               
                 K-12 
                 F −  λ −  rph-1 INV(rrnD, rrnE) 
                 Jer sen, 1993 
               
             
          
           
               
                 Plasmids 
               
             
          
           
               
                 pBRI-cymR1 
                 pBRI80 plasmid containing one copy 
                 Choi, et al., 2006 
               
               
                   
                 of cymR expression cassette 
               
               
                 pNEW-pre 
                 pET36 plasmid containing P km -cymR 
                 This study 
               
               
                   
                 expression cassettes, lack of T7 
               
               
                   
                 promoter and lac operator 
               
               
                 pCR2.1-TOPO 
                 PCR cloning vector 
                 Invitrogen Inc. 
               
               
                 pCR-P km -cymR 
                 pCR2.1-TOPO plasmid containing 
                 This study 
               
               
                   
                 P km -cymR 
               
               
                 PCR-T5OP 
                 pCR2.1-TOPO plasmid containing 
                 This study 
               
               
                   
                 P T5 -operator 
               
               
                 pCR-bgl 
                 pCR2.1-TOPO plasmid containing bgl 
                 This study 
               
               
                 pCR-est 
                 pCR2.1-TOPO plasmid containing estl 
                 This study 
               
               
                 pCR-PhaC1 or C2 
                 pCR2.1-TOPO plasmid containing 
                 This study 
               
               
                   
                 phaC1 or C2 
               
               
                 pNEW 
                 Newly constructed regulative 
                 This study 
               
               
                   
                 expression vector 
               
               
                 pNEW-mfh 
                 pNEW vector containing mfh fusion 
                 This study 
               
               
                   
                 peptide expression cassette 
               
               
                 pNEW-phaC1 or 2 
                 pNEW vector containing PhaC1 or C2 
                 This study 
               
               
                   
                 expression cassette 
               
               
                 pNEW-bgl 
                 pNEW vector containing lactase 
                 This study 
               
               
                   
                 expression cassette 
               
               
                 pNEW-est 
                 pNEW vector containing esterase 
                 This study 
               
               
                   
                 expression cassette 
               
               
                 pNEW-gfp 
                 pNEW vector containing gfp 
                 This study 
               
               
                   
                 expression cassette 
               
               
                 pET36(b) 
                 T7 based expression vector 
                 Novagen 
               
               
                 pCESTa 
                 Esterase gene source 
                 Choi et al., 2004 
               
               
                 pEBIG4 
                 Lactase gene source 
                 Hung et al., 2001 
               
               
                 pTSN-6A 
                 Source of fusion peptide mfh 
                 Csborne et al., 2003 
               
               
                   
               
             
          
         
       
     
       Results: 
       [0036]    The basic mechanism of the cumate regulated gene expression in  E. coli  is depicted in  FIG. 1 .  FIG. 1  shows a schematic diagram of the mechanism of action of the cumate-switchable expression system. (a) In the absence of a cumate, inducer, the repressor protein (cymR) is bound to the operator site upstream of the reporter gene or gene of interest, and block the transcription. (b) The presence of the cumate is necessary for transcription of gene of interest. The addition of cumate rapidly alters the inactive conformation (operator-cymR), facilitating the formation of the cymR-cumate complex and detached the cymR from the operator, and activating transcription of the downstream reporter gene. The cymR-cumate complex is unable to bind to operator site. 
         [0037]    Development of regulated expression vector pNEW-gfp. To develop a new generation of tightly regulated  E. coli  expression vectors, we applied T5 promoter-cumate operator carrying vector in cooperation with cymR repressor encoding gene in the same plasmid ( FIG. 2 ). 
         [0038]    Validation of the developed expression system in  E. coli  hosts. Since T5 promoter is recognized by  E. coli  RNA polymerase, developed expression vectors can be applied to any type of  E. coli  strain, as shown in  FIG. 3 .  FIG. 3A  depicts plate assays, while  FIG. 3B  depicts liquid culture assays in culture tubes. In  FIG. 3 , the regulated expression of GFP (green fluorescent protein) in various  E. coli  strains as host is depicted. In  FIG. 3B , tube #1 contains  E. coli  DH5a, tube #2 contains  E. coli  S17-1 λ/pir, tube #3 contains  E. coli  K12, tube #4 contains  E. coli Top10, and tube #5 contains  E. coli  BL21(DE3). 
         [0039]    Heterologous gene expression. The performance or the cumate-regulated expression system was examined with various proteins as reporter. 
       Example 1 
     Green Fluorescent Protein (GFP) Expression 
       [0040]      FIG. 4  depicts a comparison between T7 system and cumate system for GFP expression in plates containing IPTG (1 mM) and cumate (0.12 mM) as inducer, respectively. It is evident from  FIG. 4  that the cumate systems dramatically outperforms the IPTG system for expressing GFP in host cells. 
       Example 2 
     Expression of Polyhydroxyalkanoic Acids Synthetase (PhaC1 and PhaC2) Genes in  E. coli  Top10 
       [0041]    Genes encoding PhaC1 and C2 were amplified from  Pseudomonas fluorescens  GK13 and cloned into  E. coli  Top10 using cumate expression system. Amplified genes were successfully expressed in  E. coli  Top 10, and recombinant  E. coli  Top 10 produced PHB-like granules as shown in  FIG. 5 .  FIG. 5  depicts a physical map of pNEW-PhaC1, 2 and microscopic observation of the recombinant strains upon cumate induction (0.12 mM). 
       Example 3 
     Production of Esterase Using Cumate Expression System in  E. coli    
       [0042]      FIG. 6  depicts heterologous gene expression of esterase in  E. coli  Top10 using the cumate expression system of the present invention. Recombinant strain was streaked on the plate containing 1% (v/v) tributyrin as a substrate of esterase without and with cumate (0.12 mM) as an inducer, respectively. It is evident from  FIG. 6  that the cumate expression system was successful at heterologous gene expression of esterase. 
       Example 4 
     Production of Beta-Galactosidase Using Cumate Expression System in  E. coli  Top 10 
       [0043]      FIG. 7  depicts the expression profile of recombinant β-galactosidase on SDS-PAGE. Lane M is protein standard marker. Lane 1 is the first eluted sample as purified β-galactosidase using Ni-NTA mini affinity column. Lane 2 is the second eluted sample from the same column as Lane 1. Lanes 3 and 4 are crude protein samples 1 and 3 hr after induction, respectively. It is evident from  FIG. 7  that β-galactosidase has been successfully expressed in  E. coli  Top 10 by the cumate expression system of the present invention. 
       Example 5 
     Production of Synthetic Thrombin Inhibitor Peptide Using Carrier Protein (SFC120) to Form Fusion Peptide in  E. coli    
       [0044]      FIG. 8  depicts the SDS-PAGE profile of soluble and insoluble fractions. Fusion peptide was produced in the form of inclusion body as expected, and the yield of fusion peptide reached about 85% of total cellular protein. 
       Example 6 
     Bench Top Fermentation 
       [0045]      FIG. 9A  is a photograph of a fermenter with sterilized  E. coli  cultivation medium to show the original color of the cultivation medium. The original color is a gray/brown. 
         [0046]      FIG. 9B  is a photograph of two fermenters side-by-side, each fermenter containing cultivation medium and  E. coli  cells transformed with GFP. The fermenter on the left has the T7 expression system with no IPTG added yet. The fermenter in the right has the cumate expression system of the present invention with no cumate added yet. These photographs depict the cultures prior to induction by IPTG or cumate. The color of the cultures in each fermenter is the same, a light yellow/brown. 
         [0047]      FIG. 9C  is a photograph of the fermenters depicted in  FIG. 9B  at a time 45 minutes post induction with 100 μm IPTG (T7 system) and 100 μm cumate (cumate system). GFP yields are similar at this stage. The GFP yield for the IPTG induced system is 27 mg/g. The GFP yield for the cumate induced system is 30 mg/g. The color is a brighter yellow/green than in  FIG. 9B . 
         [0048]      FIG. 9D  is a photograph of the fermenters depicted in  FIG. 9B  at a time 1 hour post induction with 100 μm IPTG (T7 system) and 100 μm cumate (cumate system). GFP yields remain similar. The GFP yield for the IPTG induced system is 37 mg/g. The GFP yield for the cumate induced system is 38 mg/g. The color is a brighter yellow/green than in  FIG. 9C . 
         [0049]      FIG. 9E  is a photograph of the fermenters depicted in  FIG. 9B  at a time 2 hours post induction with 100 μm IPTG (T7 system) and 100 μm cumate (cumate system). At this point, GFP yields begin to differ that the cumate induced culture showing better yield. The GFP yield for the IPTG induced system is 74 mg/g. The GFP yield for the cumate induced system is 84 mg/g. The color is green and brighter than the colors in  FIG. 9C . The medium in the fermenter with the cumate system is brighter green than the medium in the T7 system. 
         [0050]      FIG. 9F  is a photograph of the fermenters depicted in  FIG. 9B  at a time 3 hours post induction with 100 μm IPTG (T7 system) and 100 μm cumate (cumate system). GFP yield of the cumate induced culture is markedly greater than the IPTG induced culture. The GFP yield for the IPTG induced system is 90 mg/g. The GFP yield for the cumate induced system is 123 mg/g. The color is even brighter green than in  FIG. 9E  and the cumate induced system is brighter green than the IPTG induced system. 
         [0051]      FIGS. 10A and 10B  are graphs depicting the time course of GFP yield comparing the T7 system to the cumate system at different concentrations of inducers. For  FIG. 10A , the concentration of inducer was 100 μm, while for  FIG. 10B  the concentration of inducer was 1000 μm. After 4 hours post induction, the IPTG induced GFP expression reached its maximum, whereas the cumate induced GFP expression continues even after 8 hours post induction (see also Tables 2 &amp; 3). A similar phenomenon occurs when the cultures are induced with 1000 μM IPTG or cumate. The cumate induced GFP yield is more than double that of the IPTG induced culture. Furthermore, in cultures induced with 100 or 1000 μM cumate, expression of the GFP continues even though the culture has reached the stationary phase of growth. In other words, it is a form of resting cell GFP expression. The cumate induced culture remains healthy, no lysis occurred and no foaming was observed in contrast to the IPTG induced culture which after 8 hours post induction quickly began to lyse and GFP was released onto the culture medium. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Inducer 
                   
                   
               
               
                 conc. 
                   
                 Induction Time (h) 
               
             
          
           
               
                 (μM) 
                 Inducer 
                 1 
                 2 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
               
               
                   
               
             
          
           
               
                  100 
                 Cumate 
                 38 
                 84 
                 123 
                 164 
                 176 
                 165 
                 193 
                 222 
               
               
                   
                 IPTG 
                 37 
                 74 
                 90 
                 96 
                 85 
                 58 
                 68 
                 67 
               
               
                 1000 
                 Cumate 
                 36 
                 71 
                 110 
                 141 
                 149 
                 155 
                 249 
                 289 
               
               
                   
                 IPTG 
                 37 
                 60 
                 83 
                 103 
                 118 
                 135 
                 145 
                 — 
               
               
                   
               
             
          
         
       
     
         [0052]    Table 2 shows results for the specific yield of GFP (mg/g ×) up to 8 hours of induction for T7 and cumate expression systems in  E. coli  BL21(DE3)pLysS for two inducer concentrations. All results obtained were in defined medium A. The value ‘x’ is dry weight in g/L. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Inducer conc. 
                   
                 Induction Time (h) 
               
             
          
           
               
                 (μM) 
                 Inducer 
                 1 
                 2 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
               
               
                   
               
             
          
           
               
                  100 
                 Cumate 
                 602 
                  644 
                 3002 
                 4719 
                 5443 
                 6194 
                 6977 
                 7838 
               
               
                   
                 IPTG 
                 554 
                 1300 
                 1778 
                 2486 
                 2035 
                 1567 
                 1948 
                 2090 
               
               
                 1000 
                 Cumate 
                 486 
                 1459 
                 2851 
                 4593 
                 4885 
                 5989 
                 9666 
                 11150 
               
               
                   
                 IPTG 
                 606 
                 1191 
                 1966 
                 2464 
                 3340 
                 4238 
                 4079 
                 — 
               
               
                   
               
             
          
         
       
     
         [0053]    Table 3 shows results for the total yield of GFP (mg/L) up to 8 hours of induction for T7 and cumate expression systems in  E. coli  BL21(DE3)pLysS for two inducer concentrations. All results obtained were in defined medium A. 
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         [0089]    Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.