Balancing the Production of Two Recombinant Proteins inEscherichia coliby Manipulating Plasmid Copy Number: High-Level Expression of Heterodimeric Ras Farnesyltransferase

The native Ras farnesyltransferase heterodimer (αβ) and a heterodimer with a truncated α subunit (α′β) were overproduced at a high level and in a soluble form inEscherichia coli.The α, α′, and β subunits were synthesized from individual plasmid vectors under the control of bacteriophage T7 promoters. Although each subunit could be expressed at a high level by itself, when either the α or α′ and the β plasmid were present in cells at the same time, the α and α′ subunits were preferentially expressed to such a degree that little or none of the β subunit accumulated. A satisfactory balance between both combinations of subunits (αβ and α′β) was achieved by making incremental adjustments in the copy number of the β-encoding plasmid. As the copy number of the β plasmid increased, so did the ratio of β:α or β:α′, but there was little difference in the total amount of recombinant protein (α + β or α′ + β) that was produced. This may be a generally useful method for balancing the production of two recombinant polypeptides inE. coli.A noteworthy advantage of this approach is that it can be undertaken without first determining the cause of the imbalance.     

Heterologous Expression inEscherichia coliof Soluble Active-Site Random Mutants of Haloalkane Dehalogenase fromXanthobacter autotrophicusGJ10 by Coexpression of Molecular Chaperonins GroEL/ES

A system for heterologous expression inEscherichia coliof dehaloalkane dehalogenase DhlA fromXanthobacter autotrophicusstrain GJ10 is presented. The strategy involved overexpression ofE. colichaperonins GroEL/ES which facilitated the production of soluble DhlA. When active-site mutant forms were constructed they could not to any detectable degree be expressed in a soluble state in the absence of overproduced GroEL/ES. However, with the described expression system, wild-type DhlA as well as variant forms randomly mutated in the active-site residues Phe172 and Trp175 were reliably produced. An introduced C-terminal (His)₅-tag provided an immunological handle as well as a site for metal ion coordination utilized in affinity chromatography for the purification of recombinant DhlA. The purified His-tagged enzyme, DhlA-5His, was confirmed to be catalytically fully active when measuring the dehalogenase activity with dichloroethane as substrate.     

Cloning of cDNA Encoding Rat Spermatidal Protein TP2 and Expression inEscherichia coli

Rat spermatidal protein TP2 is a basic nuclear protein containing two atoms of zinc bound per molecule. We report here cloning of complementary DNA encoding rat TP2 by the RT-PCR method. The nucleotide sequence of cloned TP2 cDNA differs at a few positions from the sequence already reported in the literature. We have cloned rat TP2 cDNA into the expression vector pTrc 99A. Upon induction with 1 mMIPTG, there was a low level of expression of TP2 which could be recovered in the soluble form. Recombinant TP2 was purified from the soluble extract ofE. coliusing nickel–agarose and heparin–agarose chromatography and was shown to be identical to native rat TP2 as revealed by immunoblotting with anti-rat TP2 antibodies and radioactive⁶⁵Zn-blotting.     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.     

Identification of DNA topoisomerases involved in immediate and transient DNA relaxation induced by heat shock inEscherichia coli

The linking number of plasmid DNA in exponentially growingEscherichia coli increases immediately and transiently after heat shock. The purpose of this study was to search for DNA topoisomerases that catalyze this relaxation of DNA. Neither introduction of atopA deletion mutation nor treatment of cells with DNA gyrase inhibitors affected the DNA relaxation induced by heat shock. Thus, DNA topoisomerase I and DNA gyrase are apparently not involved in the process. However, the reaction was inhibited by nalidixic acid or by oxolinic acid in thetopA mutant and the reaction was resistant to nalidixic acid in atopA mutant carrying, in addition, thenalA26 mutation. These results are interpreted as indicating that both DNA topoisomerase I and DNA gyrase are involved in the DNA relaxation induced by heat shock.     

High-Level Expression of Soluble Protein inEscherichia coliUsing a His6-Tag and Maltose-Binding-Protein Double-Affinity Fusion System

Using the maltose-binding protein (MBP) fusion vector pMAL-c1 from C. V. Mainaet al.(1988,Gene74, 365–373), we have constructed expression vectors which contain a sequence encoding six consecutive histidine residues (His₆-tag) at the 3′ end of the MBP-encoding malE gene which is followed by either a thrombin-binding site (LVPRGS) or a factor Xa-binding site (IEGR). The benefits of this approach include: (a) high expression levels of soluble MBP fusion proteins (exceeding 2% of the total cellular protein), (b) high-quality purification of proteins under various conditions (high salt, low salt, denaturing, nondenaturing, etc.), and (c) two alternative protease cleavage sites to test for the most efficient cleavage of each fusion protein. We also constructed these MBP–His₆-tag expression vectors with alternative selection markers (Amp^r, Kan^r) and alternative promoters (tac, T7). Using these constructs, we expressed and purified several proteins of which we present two, penicillin-binding protein PBP2a and UDP-N-acetylmuramate:l-alanine ligase (MurC), and compare their expression level and purity with other expression systems. We also discuss the use of minimal media with supplements versus rich media and cell growth strategies to optimize the protein yield in general and for isotope labeling.     

Requirement of DNA Gyrase for the initiation of chromosome replication in Escherichia coli K-12

Summary It has been found that strains carrying mutations in the dnaA gene are unusually sensitive to COU, NAL or NOV, which are known to inhibit DNA gyrase activities. The delay in the initiation of chromosome replication after COU treatment has been observed in cells with chromosomes synchronized by amino acid starvation or by temperature shift-up (dnaA46). The unusual sensitivity of growth to COU of the initiation mutant runs parallel to a higher sensitivity to the drug of the initiation of chromosome replication.The double mutant, dnaA46 cou-110 has been isolated and mutation cou-110 conferring resistance of growth, initiation and elongation of chromosome replication to COU was mapped in the gene coding for the subunit of DNA gyrase. The reduced frequency of appearance of the mutants resistant to COU, NAL or NOV in the initiation mutant suggests that some mutations in genes coding for DNA gyrase subunits cannot coexist with the dnaA46 mutation. The possible mechanisms of the requirement of DNA gyrase for dnaA-dependent initiation of E. coli chromosome are discussed.Abbreviations used COU coumermycin A₁ - NAL nalidixic acid - NOV novobiocin     

Plasmid mediated alterations in composition and structure of envelopes of Escherichia coli B/r

Seven transmissible (Tra⁺), antibiotic resistance (r) plasmids, which confer sensitivity to the filamentous bacteriophage IKe on an Escherichia coli B/r strain harboring them, have been examined for the changes they evoke in the host cell membranes. The plasmids rR48and rR269, like rRM98 [1], cause a significant reduction in the density of the outer membrane and the virtual elimination of its 36 500 dalton protein. These 2 properties do not appear to be altered when rR45, rR199 or rR205 are the resident plasmids. No changes in the inner membrane proteins are seen in any of these strains. In the case of the rR46-bearing strain, the density of the outer membrane is increased and the level of the 36 500 dalton protein is unaltered; in addition, several changes in both inner and outer membrane proteins are seen. Spontaneous IKe resistant mutants isolated from strains lacking the 36 500 dalton protein are either Tra⁺ or Tra^?. Since most of them also lack this protein, the latter is not important to the expression of inter-bacterial gene transfer and IKe sensitivity.On freeze fracture, strains lacking the 36 500 dalton protein cleave almost exclusively within the outer membrane. The plasmidless host and the remaining plasmid-bearing strains show a strong fracture plane within the cytoplasmic membrane. Despite the fact that most of the plasmid-bearing strains used are proficient in effecting interbacterial plasmid transfer, no morphological differences which can be correlated with this function have been observed between their etched cell surfaces and that of the plasmidless host.     

Isolation and identification of 5-methylthioribose from Escherichia coli B

5-Methylthioribose was isolated after incubation of Escherichia coli B in a glucose-salts medium. At least 60% of the radioactivity in absolute ethanol extracts of the residue from lyophilized medium supplemented with ³⁵SO₄²⁻ was located in two chromatographic areas that were identified as 5-methylthioribose and its sulfoxide. The sulfoxide was formed by oxidation of 5-methylthioribose during necessary processing of cultures and fractions. These compounds were characterized by functional group analysis and chromatographic comparison with authentic material. 5-Methylthioribose sulfoxide was isolated from 12 l of incubation medium of E. coli. After purification in three paper and one thin-layer chromatographic systems, 50 μg was obtained. The trimethylsilyl derivative of this compound was compared with that of authentic 5-methylthioribose sulfoxide. Gas chromatography and mass spectrometry confirmed the identity. This is the first report of 5-methylthioribose from a bacterium.     

Conformational Dynamics of the Escherichia coli DNA Polymerase Manager Proteins UmuD and UmuD′

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD′. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent − 1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD′₂C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, which is noncleavable but is a partial biological mimic of the cleaved form UmuD′, has been identified. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD′, and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD′, is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD′ and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD′ because, in both cases, the arms are relatively unbound from the globular domain. Gas-phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter timescales than solution experiments, show that UmuD′ incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is of critical importance for their many protein interactions.     

Conjugal Transfer of Plasmid DNA fromEscherichia colito Enterococci: A Method to Make Insertion Mutations

Shuttle vector pAT18 was transferred by conjugation fromEscherichia coliS17-1 toEnterococcus faecalisOG1RF andEnterococcus faeciumSE34. Transfer was mediated by the transfer functions of plasmid RK2 inE. coliS17-1 and the origin of conjugal transfer (oriT) located on pAT18. TheoriTsequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene ofE. faecalisOG1RF was cloned into pTEX5235 and transferred by conjugation fromE. coliS17-1 toE. faecalisOG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster fromE. faecalis,with a transposon insertion inpyrC,was also transferred fromE. coliS17-1 toE. faecalisOG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in apyrCknockout mutant showing an auxotrophic phenotype.     

Cloning and sequencing of two genes fromStaphylococcus carnosus coding for glucose-specific PTS and their expression inEscherichia coliK-12

Phosphoenolpyruvate (PEP)-dependent phosphorylation experiments have indicated that the grampositive bacteriumStaphylococcus carnosus possesses an EIICBA fusion protein specific for glucose. Here we report the cloning of a 7 kb genomic DNA fragment containing two genes,glcA andglcB, coding for the glucose-specific PTS transporters EII^Glc1 and EII^Glc2 which are 69% identical. The translation products derived from the nucleotide sequence consist of 675 and 692 amino acid residues and have calculated molecular weights of 73 025 and 75 256, respectively. Both genes can be stably maintained inEscherichia coli cells and restore the ability to ferment glucose toptsG deletion mutants ofE. coli. This demonstrates the ability of the PTS proteins HPr and/or EIIA^Glc of a gram-negative organism (E. coli) to phosphorylate an EIICBA^Glc from a gram-positive organism (S. carnosus).     

Expression, Purification, and Characterization of RecombinantEscherichia coliPyridoxine 5′-Phosphate Oxidase

A previously clonedpdxHgene fromEscherichia colicoding for pyridoxine 5′-phosphate oxidase was transferred to a pET22b vector and expressed inE. coliHMS174(DE3) cells. The soluble overexpressed enzyme was rapidly purified in high yield using two chromatography columns with an overall purification of about 2.8-fold. The purified enzyme contained tightly bound FMN. The enzyme exhibited the same spectral properties and similar kinetic constants to those previously reported by G. Zhao and M. E.Winkler (J. Bacteriol.177, 883, 1995), but differed from the properties reported by other investigators. A rapid procedure was developed for preparing apoPNP Ox in high yield. Both the holo- and apoenzymes were homodimers. The molar absorbtivity coefficient for the protein was determined for the fully active apoPNP Ox from is amino acid composition. Using this value and the spectral properties of the bound FMN it was shown by three different methods that the dimeric enzyme contains two molecules of bound FMN per dimer and not one FMN as previously reported.     

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins inEscherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor ofEscherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of thetac promoter, and LetD protein was induced by adding isopropyl-d-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [³⁵S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of ³². We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of ³².     

APPswe基因慢病毒表达质粒的构建及对其SH-SY5Y细胞生长和凋亡的影响

为考察APPswe基因的表达对细胞生长和凋亡作用的影响,本研究采用gateway分子重组技术构建表达APPswe基因的慢病毒质粒,并将该质粒转染至SH-SY5Y细胞中.用RT-PCR和Western blot技术分别测定APPswe基因的mRNA转录和蛋白翻译水平.活细胞计数法和细胞外乳酸脱氢酶法分别测定细胞存活力和细胞膜损伤程度.Annexin V-FITC/PI染色流式细胞术测定细胞凋亡水平,Hoescht 33342染色观察细胞核形态变化,DCFH-DA染色法测定胞内活性氧水平.转录和翻译水平的验证表明,APPswe基因能够在SH-SY5Y细胞中相对稳定地表达.表达APPswe基因后,细胞的存活能力降低,损伤程度增加,细胞内活性氧水平上升,细胞核浓缩聚集,细胞发生凋亡反应.试验结果表明,APPswe基因的表达对SH-SY5Y细胞生长产生抑制作用,造成细胞损伤和凋亡,表达APPswe基因的SH-SY5Y细胞系可应用于体外AD病理发生机制和药物作用的研究.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Isolation of Escherichia coli mRNA and Comparison of Expression Using mRNA and Total RNA on DNA Microarrays

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3′-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.     

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB_69-333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB_69-333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB_69-333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.