Heterologous protein expression using a novel stress-responsive protein of E. coli RpoA as fusion expression partner

E. coli proteome response to the stressor 2-HEDS was analyzed through two-dimensional gel electrophoresis (2-DE), and we identified DNA-directed RNA polymerase α-subunit (RpoA) as stress-responsive protein. Even under stress situation where the total number of soluble proteins decreased by 9.8%, the synthesis level of RpoA was increased 1.5-fold. As a fusion expression partner as well as solubility enhancer, RpoA facilitated the folding and increased significantly the solubility of many aggregation-prone heterologous proteins (human minipro-insulin, human epidermal growth factor, human prepro-ghrelin, human interleukin-2, human activation induced cytidine deaminase, human glutamate decarboxylase, Pseudomonas putida cutinase, human ferritin light chain, human granulocyte colony-stimulating factor, and cold inflammatory syndrome1 protein Nacht domain) in E. coli cytoplasm. Due probably to intrinsic high folding efficiency and/or chaperone-like activity, RpoA was very effective in shielding interactive surfaces of heterologous proteins that are associated with non-specific protein–protein interaction leading to the formation of inclusion bodies. RpoA was also well suited for the production of biologically active fusion mutant of Pseudomonas putida cutinase that is of much biotechnological and commercial interest.     

Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression

The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD(448-585)), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.     

Expression of the measles virus nucleoprotein gene in Escherichia coli and assembly of nucleocapsid-like structures

To investigate the use of fusion systems to aid the purification of recombinant proteins for structure/function studies and potential uses as diagnostic reagents, the measles virus (MV) gene encoding the nucleoprotein was cloned and expressed in Escherichia coli in three forms: as a full-length intact protein and as two fusion proteins. Expression of the intact N gene under the control of the tac promoter in the pTrc99c plasmid produced a protein of the correct size (60 kDa) which represented approx. 4% of the total cellular protein, and was recognised by known measles positive human sera. ‘Herringbone’ structures characteristic of paramyxovirus nucleocapsids (NuC) were identified in fractured cells examined by electron microscopy. The production of NuC-like structures in a prokaryotic cell indicates folding of the nucleoprotein can occur in the absence of MV genomic RNA, other MV-encoded gene products and eukaryotic cell proteins or RNA, to produce structures which are morphologically and antigenically similar to those seen in virus-infected cells. Conversely, synthesis of N protein as a fusion protein with either E. coli β-galactosidase or the E. coli maltose-binding protein resulted in the production of fused proteins which could not be assembled into NuC-like structures or readily used as diagnostic reagents. However, the ability of MV N protein to form NuC-like structures in E. coli will facilitate structure/function and mutational analysis of the NuC protein.     

Biocatalytic synthesis of monocyclic arene-dihydrodiols and -diols by Escherichia coli cells expressing hybrid toluene/biphenyl dioxygenase and dihydrodiol dehydrogenase genes

The hybrid toluene/biphenyl dioxygenase, which is encoded by the todC1 gene of Pseudomonas putida F1 and the bphA2A3A4 genes of Pseudomonas pseudoalcaligenes KF707, has substrate ranges wider than toluene dioxygenase endoced by the todC1C2BA genes of P. putida F1. We carried out growing cell reactions by Escherichia coli expressing the todC1-bphA2A3A4 genes for the comprehensive production of monocyclic arene-dihydrodiols. As a result, we successfully biotranformed acetophenone-related compounds (acetophenone, propiophenone, and butyrophenone) to the corresponding cis-dihydrodiols. Furthermore, we performed the bioconversion experiments by E. coli cells expressing the bphB (dihydrodiol dehydrogenase) gene in addition to todC1-bphA2A3A4 to produce a series of monocyclic arene-diols. Consequently, toluene, benzene, stylene, p-xylene, acetophenone, propiophenone, butyrophenone, and trifluoroacetophenone were converted to the corresponding vicinal diols. The antioxidative activity of these generated diol compounds was markedly higher than that of the substrate used.     

Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

Killing of target cells by redirected granzyme B in the absence of perforin

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 °C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.     

A novel Escherichia coli solubility enhancer protein for fusion expression of aggregation-prone heterologous proteins

Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli.     

Surface display of a glucose binding protein

Glucose binding protein (GBP) from Escherichia coli has been widely used to develop minimally invasive glucose biosensors for diabetics. To develop a cell-based glucose biosensor, it is essential to functionally display GBP on the cell surface. In this study, we designed a molecular structure to display GBP on the outer membrane of E. coli. We fused GBP with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli). With this molecular design, we have successfully displayed GBP on the surface of E. coli. Using FITC-conjugated Dextran, we demonstrated that glucose’s binding sites of surface-displayed GBP were accessible to glucose. Furthermore, we showed that glucose transport in a GBP-deficient E. coli NM303 could be restored by displaying GBP on the surface of NM303. 0.51 h⁻¹ of specific growth rate was attained for NM303/pESDG grown in M9 minimal medium supplemented with 2 g/l glucose, whereas no growth was observed for NM303 in the same medium. Both NM303 and NM303/pESDG grew in M9 medium supplemented with 1 mM of fucose. Because cell surface is an interface between intracellular and extracellular molecular events, this technique paves a way to develop cell-based glucose biosensors.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> malate dehydrogenase,a novel solubility enhancer for heterologous proteins synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.     

A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

Phospholipids of Five Pseudomonad Archetypes for Different Toluene Degradation Pathways

Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was used to determine intact phospholipid profiles for five reference pseudomonad strains harboring different (aerobic) toluene catabolic pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. These five strains contained a predominant pool of phosphatidylethanolamines. Other phospholipids identified include phosphatidylglycerol, phosphatidylserine, phosphatidylmethylethanolamine, and phosphatidyldimethylethanolamine. There was a clear separation in phospholipid profiles that allows for the differentiation between the Pseudomonas and Burkholderia genera. Factor analysis of the phospholipid profiles showed that B. cepacia G4, P. putida mt-2, and B. pickettii PKO1 were clearly separated, while P. putida F1 and P. mendocina KR1 were clustered as a group. These results suggest that intact phospholipid profiling could be used to evaluate the relative abundance of specific degraders in bioreactors or in aquifer material. Nevertheless, the usefulness of this technique for taxonomic characterization of such complex samples remains to be demonstrated because of potential confounding effects of overlapping profiles and potential changes in phospholipid composition due to different growth conditions.     

Microbiological transformations 52.: Biocatalysed Baeyer–Villiger oxidation of 1-indanone derivatives

The microbiological Baeyer–Villiger oxidation of various substituted 1-indanones is described. Three bacterial strains have been explored: an E. coli TOP10 [pQR 239] constructed to overexpress the cyclohexanone monoxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871, an E. coli TOP10 [hapE] strain recently constructed to overexpress 4-hydroxyacetophenone monoxygenase (HAPMO) of Pseudomonas fluorescens ACB and the wild type Pseudomonas sp. NCIMB 9872 strain known to metabolise cyclopentanone. This last strain oxidised some of the proposed substrates, leading to the corresponding lactones with good to excellent yields depending on the aromatic ring substituents.     

Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition.

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins

We have established a eukaryotic protein expression and purification system by using the yeast Schizosaccharomyces pombe as the host and the glutathione S-transferase (GST) as a protein purification tag. This system provides opportunities for rapid, inexpensive, and high yield production of proteins in a eukaryotic organism. Unlike E. coli, S. pombe provides for post-translational modifications of the proteins, which are often critical for the structure and function of eukaryotic proteins. Two vectors have been constructed for protein expression in S. pombe, pESP-1 and pESP-2. Both vectors use the nmt1 promoter for constitutive or induced expression of the gene of interest. Expressed GST-tagged proteins are easily and rapidly purified using glutathione agarose beads. The GST tag can be removed from the fusion proteins by treatment with either the thrombin or enterokinase protease. Proteins expressed from the pESP-2 vector will yield native amino acid sequence when the GST tag is removed by treatment with enterokinase. Nine proteins have been purified by using the system with yields ranging from 1.0 mg/l to 12.5 mg/l of induced culture.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Catalytic domain of Salmonella typhimurium 2-oxoglutarate dehydrogenase is localized in N-terminal region

2-Oxoglutarate dehydrogenase (lipoamide) [OGDH or E1o: 2-oxoglutarate: lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinating); EC 1.2.4.2] is a component enzyme of the 2-oxoglutarate dehydrogenase complex. Salmonella typhimurium gene encoding OGDH (ogdh) has been cloned in Escherichia coli. The libraries were screened for the expression of OGDH by complementing the gene in E. coli E1o-deficient mutant. Three positive clones (named Odh-3, Odh-5 and Odh-7) contained the identical 2.9 kb Sau3AI fragment as determined by restriction mapping and Southern hybridization, and expressed OGDH efficiently and constitutively using its own promoter in the heterologous host. This gene spans 2878 bases and contains an open reading frame of 2802 nucleotides encoding a mature protein of 927 amino acid residues (M_r=110,000). The comparison of the deduced amino acid sequence of the cloned OGDH with E. coli OGDH shows 91% sequence identity. To localize the catalytic domain responsible for E. coli E1o-complementation, several deletion mutants lacking each portion of the ogdh gene were constructed using restriction enzymes. From the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a polypeptide which showed a complementation activity with an M_r of 30,000 was detected. The catalytic domain was localized in N-terminal region of the gene. Therefore, this is a first identification of the catalytic domain in bacterial ogdh gene.