Control Of T7 Promoter Research Articles

EXPRESSION AND PURIFICATION OF RECOMBINANT OUTER SURFACE PROTEIN D OF BORRELIA BURGDORFERI

Summary To carry out the immunological experiments on the serum of Multiple Sclerosis (MS) patients, based on a correlation between Borrelia burgdorferi infection and contracting MS autoimmune disease the outer surface protein D (OspD) of the bacterium was expressed and purified. A clone containing the OspD gene in pET11a expression vector under the control of T7 promoter was transformed to the bacterial host BL21 (DE3). Some of the colonies were selected for IPTG-induced expression. The colony with the highest amount of OspD was selected for large-scale expression. Large-scale protein purification was performed by the reversed phase HPLC with a C4 preparative column; ultimately, the expressed purified protein was confirmed by the Western blot technique.

Solubility, immunogenicity and physical properties of the nucleocapsid protein of Nipah virus produced in Escherichia coli.

The nucleocapsid (N) protein of Nipah virus (NiV) can be produced in three Escherichia coli strains [TOP10, BL21(DE3) and SG935] under the control of trc promoter. However, most of the product existed in the form of insoluble inclusion bodies. There was no improvement in the solubility of the product when this protein was placed under the control of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21(DE3) cell cultures. Solubility analysis of N- and C-terminally deleted mutants revealed that the full-length N protein has the highest solubility. The soluble N protein could be purified efficiently by sucrose gradient centrifugation and nickel affinity chromatography. Electron microscopic analysis of the purified product revealed that the N protein assembled into herringbone-like particles of different lengths. The C-terminal end of the N protein contains the major antigenic region when probed with antisera from humans and pigs infected naturally.

Construction, expression, and characterization of a recombinant annexin B1-low molecular weight urokinase chimera in Escherichia coli.

To produce a thrombi-targeting plasminogen activator, low molecular weight single-chain urokinase gene (scuPA32k) was spliced with the full-length cDNA of annexin B1 gene (anxB1) by overlap extension method. The fused gene anxB1scuPA was ligated into pET28a vector, transformed into E. coli BL21-RIL, and then induced to express under the control of T7 promoter. The AnxB1ScuPA protein expressed amounted to 22% of the total bacterial proteins. The product was refolded, and then purified by using DEAE Sepharose fast flow ion-exchange column and Superdex S-200 gel-filtration column. HPLC analysis revealed that the final purity is about 95%. The specific activity of AnxB1ScuPA, measured as amidolytic activity, reached 100,000 IU/mg. It had a similar S2444 catalytic efficiency (kcat/Km) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chimera fully retained the components of enzymatic and membrane-binding activities of the parent molecules. In vivo test revealed that, the dogs administered with AnxB1ScuPA had less reperfusion time, higher reperfusion ratio, and less bleeding effects than those with urokinase. These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.

대장균에서 chaperons 분자와 저온배양에 의한 CGTase의 가용성 발현 증대

E. coli에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현과 저온 배양의 공동작용 효과에 대해 조사하였다. 실험에 사용된 cgt와 groEL/ES 유전자를 발현하는 pTCGTl과 pGroll은 각각 T7 promoter와 Pzt-1 promoter에 의해 조절되고 이들을 E. coli에 도입시켰다. 대수증식기 초기(2 hr)와 대수증식기 중기(3 hr)에 tetrarycline 10 ng/ml 과 IPTG 1 mM을 첨가하여 각각의 유전자를 발현시켰다. CGTase활성과 specific artivity 측정 시 <TEX>$37^{\circ}C$</TEX>에서 pTCGTl 단독 발현 보다 <TEX>$25^{\circ}C$</TEX>에서 chaperone과 함께 발현시킨 경우 2배나 높은 활성이 측정됐으며, SDS-PACE 분석결과 <TEX>$37^{\circ}C$</TEX>에서 단독 발현 시킬경우 20% 정도 가용성 형태로 발현되던 것이 <TEX>$25^{\circ}C$</TEX>에서 chaperone과 공발현 시에는 거의 3.5배가 넘는 69%가 가용성으로 전환됨을 알 수 있었다. 이와 같이 분자 chaperone과 <TEX>$25^{\circ}C$</TEX>에서의 저온 배양은 E. coli에서 활성형질 가용성 CGTase의 생산 증가에 큰 영향을 미치는 것으로 나타났다. The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at <TEX>$25^{\circ}C$</TEX> with groEL/ES expression improved the activity of CGTase by two fold, compared to <TEX>$37^{\circ}C$</TEX> cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at<TEX>$25^{\circ}C$</TEX>, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at <TEX>$37^{\circ}C$</TEX> without chaperone. The amount of soluble CGTase from <TEX>$25^{\circ}C$</TEX> culture with chaperone was 3.5-fold higher than that of <TEX>$37^{\circ}C$</TEX> culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.

Biochemical Investigations of Control of Replication Initiation of Plasmid R6K

The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.

N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

The L protein (241 kD) of vesicular stomatitis virus (VSV) is the most important subunit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein (GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kept on the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in the perinuclear regions. And the fusion protein with 96–120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108–120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.

Biotin–ubiquitin tagging of mammalian proteins in Escherichia coli

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin–ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin–ubiquitin tag from biotin–ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.

Ferredoxin reductase enhances heterologously expressed cytochrome CYP105D1 in Escherichia coli and Streptomyces lividans

The ability of two different ferredoxin reductases from Streptomyces coelicolor, to enhance the amount of active recombinant Streptomyces griseus soyC (CYP105D1) was investigated in both Escherichia coli and Streptomyces lividans. In E. coli a two-plasmid system and a single operon construct were used for expression of the CYP105D1 and the ferredoxin reductase(s) under the control of T7 promoters. Expression levels of CYP105D1 were found to range between 85 and 280 nmol l −1 cell culture after prolonged growth. In S. lividans the CYP105D1 and its ferredoxin were cloned downstream of the Pact1 promoter in the E. coli/ Streptomyces shuttle vector pBW160. The recombinant E. coli and S. lividans cells converted 7-ethoxycoumarin into 7-hydroxycoumarin efficiently. Expression of a ferredoxin reductase as an operon with CYP105D1 and its ferredoxin enhances the o-dealkylation of 7-ethoxycoumarin. Ferredoxin NADPH reductase was found to enhance the level of the active form of CYP105D1 monooxygenase when no substrate was present.

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro.

To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG-AGT) in vitro. Two different monomers of anti-mtp53 maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G(5)-A(5)) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR, pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme). Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEM-T vector under the T7 promoter control. The (32)p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with (32)p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis. In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 degrees and 25 mM MgCL(2). The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either. pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good foundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.

Regulation of gene expression by sequence-specific alkylating polyamide.

In order to investigate the inhibition of gene expression by a new type of hairpin polyamide-CPI conjugate 1, its ability to inhibit transcription in cell free system was investigated. Sequence-selective alkylation of double-stranded DNA by 1 was investigated by denaturing gel electrophoresis using 1000 bp DNA fragment which codes for green fluorescence protein (GFP) under the control of T7 promoter. Analysis of DNA sequence indicated that 1 alkylated predominantly at the site of 5'-AGTCA-3' in coding region of GFP. The transcript by T7 RNA polymerase using the alkylated DNA as a template was analyzed by PAGE. The results clearly indicate that 1 inhibits transcription by alkylation of coding region at a nanomolar concentration.

Expression and characterization of beta-1,4-galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae.

The lgtB genes that encode beta-1,4-galactosyltransferases from Neisseria meningitidis ATCC 13102 and gonorrhoeae ATCC 31151 were isolated by a polymerase chain reaction using the pfu DNA polymerase. They were expressed under the control of lac and T7 promoters in Escherichia coli M15 and BL21 (DE3). Although the genes were efficiently expressed in E. coli M15 at 37 degrees C (33 kDa), most of the beta-1,4-galactosyltransferases that were produced were insoluble and proteolysed into enzymatically inactive polypeptides that lacked C-terminal residues (29.5 kDa and 28 kDa) during the purification steps. When the temperature of the cell growth was lowered to 25 degrees C, however, the solubility of the beta-1,4-galactosyltransferases increased substantially. A stable N-terminal his-tagged recombinant enzyme preparation could be achieved with E. coli BL21 (DE3) that expressed lgtB. Therefore, the cloned beta-1,4-galactosyltransferases were expressed under the control of the T7 promoter in E. coli BL21 (DE3), mostly to the soluble form at 25 degrees C. The proteins were easily purified to homogeneity by column chromatography using Ni-NTA resin, and were found to be active. The galactosyltransferases exhibited pH optimum at 6.5-7.0, and had an essential requirement for the Mn(+2) ions for its action. The Mg(+2) and Ca(+2) ions showed about half of the galactosyltransferase activities with the Mn(+2) ion. In the presence of the Fe(+2) ion, partial activation was observed with the beta-1,4-galactosyltransferase from N. meningitidis (64% of the enzyme activity with the Mn(+2) ion), but not from N. gonorrhoeae. On the other hand, the N(+2), Zn(+2), and Cu(+2) ions could not activate the beta-1,4- galactosyltransferase activity. The inhibited enzyme activity with the Ni(+2) ion was partially recovered with the Mn(+2) ion, but in the presence of the Fe(+2), Zn(+2), and Cu(+2) ions, the Mn(+2) ion could not activate the enzyme activities. Also, the beta-1,4-galactosyltransferase activity was 1.5-fold stimulated with the non-ionic detergent Triton X-100 (0.1-5 percent).

Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins.

Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its approximately 30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an approximately 40 nm long stalk. Taken together, our data document that the Nup84p-Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.

The Nudix hydrolases of Deinococcus radiodurans.

All 21 of the Nudix hydrolase genes from the radiation-resistant organism Deinococcus radiodurans have been cloned into vectors under the control of T7 promoters and expressed as soluble proteins in Escherichia coli. Their sizes range from 9.8 kDa (91 amino acids) to 59 kDa (548 amino acids). Two novel proteins were identified, each with two Nudix boxes in its primary structure, unique among all other known Nudix hydrolases. Extracts of each of the expressed proteins were assayed by a generalized procedure that measures the hydrolysis of nucleoside diphosphate derivatives, and several enzymatic activities were tentatively identified. In addition to representatives of known Nudix hydrolase subfamilies active on ADP-ribose, NADH, dinucleoside polyphosphates or (deoxy)nucleoside triphosphates, two new enzymes, a UDP-glucose pyrophosphatase and a CoA pyrophosphatase, were identified.

Phophoenolpyruvate carboxylase (PEPC) from a thermophilic caynobacterium, Synechococcus vulcanus

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus and expressed in E.coli BL21 (DE3) under the control of T7 promoter. The purified fusion protein (recombinant SvPEPC) showed very unusual allosteric regulatory properties. The enzyme activity was strongly activated by Fruc-1,6-P2 and slightly stimulated by Gluc-6-P, Glu and Gln. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I0.5 for Asp was increased about 2-fold by Gluc-6-P while markedly decreased about 4-fold by Fruc-1,6-P2. These allosteric properties are not readily explainable by conventional allosteric models. It seems that Fruc-1,6-P2 and Gluc-6-P do not share the same binding site on SvPEPC protein. When SvPEPC was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated in planta transformatiom under the control of the cauliflower mosaic virus 35S promoter, growth hindrance occurred on the transformants plants grown on agar plate with 1% sucrose under white light. In the early stage of growth, about 10% of plants showed bleaching in leaves or cotyledons in a gradient manner. A part of them could grow on soil but decreased apical dominance was observed in the adult plants. Leaf or cotyledon bleaching may be resulted from the exhaust of PEP due to the over-expression of SvPEPC in Arabidopsis thaliana.

Generation of an AraC-araBAD Promoter-Regulated T7 Expression System

An alternative and facile delivery system for T7 RNA polymerase has been devised and constructed. T7 gene 1 has been placed under control of the araBAD promoter element regulated by the AraC protein. Cotransformation of the resultant plasmid, pTara, with one containing a target gene under T7 promoter-regulated expression potentially allows repression by glucose and induction by arabinose in the range of 0.5 to 20 mM sugar concentration. To demonstrate the efficacy of this expression system, the p53 gene under T7 promoter control in two different plasmids was expressed in Escherichia coli using pTara as the source of T7 RNA polymerase. Repression and induction of p53 were achieved in both a lower and higher copy number plasmid, although the levels of induction were higher with the lower copy number expression vector. Cotransformation of an expression plasmid with pTara provides a low-cost method of T7 RNA polymerase-regulated expression that can be fine-tuned using glucose and arabinose concentrations to balance protein expression with potential solubility or toxicity problems.

A single amino acid substitution in the phosphoprotein of respiratory syncytial virus confers thermosensitivity in a reconstituted RNA polymerase system.

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.

Optimization of extracellular 5-aminolevulinic acid production from Escherichia coli transformed with ALA synthase gene of Bradyrhizobium japonicum

Extracellular accumulation of 5-aminolevulinic acid (ALA) by an E. coli overexpressing ALA synthase (ALAS) was achieved by inserting a hemA gene from Bradyrhizobium japonicum and expressed under the control of T7 promoter. At pH 7.0 extracellular ALA reached up to 15 mM in a jar fermenter with an addition of glycine (30 mM) and succinate (90 mM) in the medium. ALA accumulation was increased to 20 mM by adding levulinic acid (30 mM) to the cultures.

Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system.

The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus. The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.

Increased solubility of glutathione S-transferase-P16 (GST-p16) fusion protein by co-expression of chaperones groes and groel in Escherichia coli.

Human cdk (cyclin dependent kinase) inhibitor p16 was fused with glutathione S-transferase (GST) and the GST-p16 fusion protein is under the control of T7 promoter. When expressed in E. coli BL21(DE3), most products existed in the form of insoluble inclusion bodies. When co-expressed with molecular chaperones E. coli GroESL, most GST-p16 products accumulated in the soluble form with a 5-6 fold increase in solubility. When coproduced with human protein disulfide isomerase (PDI), there was no improvement in the solubility of GST-p16 fusion protein.

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.