Papers to Appear in Forthcoming Issues

Purification of Bovine S100A12 from RecombinantEscherichia coli. Kayoko Yamashita, Yuhta Oyama, Tsuyoshi Shishibori, Osamu Matsushita, Akinobu Okabe, and Ryoji Kobayashi.Kinetic Properties of Human Dopamine Sulfotransferase (SULT1A3) Expressed in Prokaryotic and Eukaryotic Systems: Comparison with the Recombinant Enzyme Purified fromEscherichia-coli. Rana Dajani, Sheila Sharp, Steven Graham, Susanne S. Bethell, Robert M. Cooke, Derek J. Jamieson, and Michael W. H. Coughtrie.Bacterial Expression and Purification of the Fab Fragment of a Monoclonal Antibody Specific for the Low-Density Lipoprotein Receptor-Binding Site of Human Apolipoprotein E. Robert Raffai, Jelena Vukmirica, Karl H. Weisgraber, Eric Rassart, Thomas L. Innerarity, and Ross Milne.Expression, Purification, and Characterization of the Recombinant Calcium-Binding Equine Lysozyme Secreted by the Filamentous FungusAspergillus niger:Comparisons with the Production of Hen and Human Lysozymes. Andrew Spencer, Ludmilla A. Morozov-Roche, Wim Noppe, Donald A. Mackenzie, David J. Jeenes, Marcel Joniau, Christopher M. Dobson, and David B. Archer.Stable, High-Level Expression of a Type I Antifreeze Protein inEscherichia coli. Robert G. Solomon and Rudi Appels.pSKAP/S: An Expression Vector for the Production of Single-Chain Fv Alkaline Phosphatase Fusion Proteins. Remko A. Griep, Charlotte van Twisk, Randolf J. Kerschbaumer, Karen Harper, Lesley Torrance, Gottfried Himmler, Jan M. van der Wolf, and Arjen Schots.Soluble Expression inEscherichia coliof Murine Endogenous Retroviral Transmembrane Envelope Protein Having Immunosuppressive Activity. Kyung Soo Kim, Ki Hwan Kim, Sung E Choi, Ji-Won Yoon, and Yup Kang.Purification of Xyloglucan-Endotransglycosylase Based on Affinity Sorption of the Active Glycosyl–Enzyme Intermediate Complex to Cellulose. Zdena Sulová and Vladimír Farkas.     

Papers to Appear in Forthcoming Issues

The Human Trifunctional Enzyme ofde NovoPurine Biosynthesis: Heterologous Expression, Purification, and Preliminary Characterization Mark T. Poch, Wen Qin, and Carol A. CaperelliIsolation and Expression of Murine Carbonic Anhydrase IV Jonathan D. Hurt, Chingkuang Tu, and Philip J. LaipisExpression of Rat Histone H1d inEscherichia coliand Its Purification M. M. Srinivas Bharath, J. R. Khadake, and M. R. S. RaoPolyethylene Glycol Conjugation of Recombinant Methioninase for Cancer Therapy Yuying Tan, Xinghua Sun, Mingxu Xu, Zili An, Xuezhong Tan, Xiuying Tan, Qinghong Han, Dusan A. Miljkovic, Meng Yang, and Robert M. HoffmanExpression inEscherichia coliof the Elongation Factor 1β Gene and Its Nucleotide T160C Mutant from the ArchaeonSulfolobus solfataricusGiuseppe Ianniciello, Mariorosario Masullo, Gennaro Raimo, Paolo Arcari, and Vincenzo BocchiniAn Expression System of Rat Calmodulin using T7 Phage Promoter inEscherichia coliNobuhiro Hayashi, Mamoru Matsubara, Akihiko Takasaki, Koiti Titani, and Hisaaki TaniguchiFunctional Expression of Secreted Mouse BST-1 in Yeast Alamgir M. M. Hussain, Hon Cheung Lee, and Chan Fong ChangEffect of Purification Protocol on the Functional Properties of Erythrocyte Membrane Protein 4.1 Ryan F. Workman and Philip S. LowAcidic Peptide-Mediated Expression of the Antimicrobial Peptide Buforin II as Tandem Repeats inEscherichia coliJae H. Lee, Il Minn, Chan B. Park, and Sun C. Kim.     

Papers to Appear in Forthcoming Issues

Preparation of Recombinant Bovine, Porcine, and Porcine W4R/R5K Leptins and Comparison of Their Activity and Immunoreactivity with Ovine, Chicken, and Human LeptinsNina Raver, Eugene E. Gussakovsky, Duane H. Keisler, Radha Krishna, Jehangir Mistry, and Arieh GertlerPurification of Histidine-Tagged Mitochondrial ADP/ATP Carrier: Influence of the Conformational States of the C-Terminal RegionChristelle Fiore, Véronique Trézéguet, Pierre Roux, Agnès Le Saux, Florence Noël, Christine Schwimmer, Delphine Arlot, Anne-Christine Dianoux, Guy J.-M. Lauquin, and Gérard BrandolinExpression and Purification of Recombinant Human Indoleamine 2,3-DioxygenaseTamantha K. Littlejohn, Osamu Takikawa, Daniel Skylas, Joanne F. Jamie, Mark J. Walker, and Roger J. W. TruscottComparative Characterization of Two Forms of Recombinant Human SPC1 Secreted from Schneider 2 CellsJean-Bernard Denault, Claude Lazure, Robert Day, and Richard LeducFunctional and Immunological Analysis of Recombinant Mouse H and L Ferritins from E. coliPaolo Santambrogio, Anna Cozzi, Sonia Levi, Ermanna Rovida, Fulvio Magni, Alberto Albertini, and Paolo ArosioExpression and Purification of Soluble and Inactive Mutant Forms of Membrane Type-1 Matrix MetalloproteinaseHeli Valtanen, Kaisa Lehti, Jouko Lohi, and Jorma Keski-OjaFunctional Human Insulin-Degrading Enzyme Can Be Expressed in BacteriaValérie Chesneau and Marsha Rich RosnerHeterologous Expression in Pseudomonas aeruginosa and Purification of the 9.2 kDa c-Type Cytochrome Subunit of p-Cresol MethylhydroxylaseCiarán N. Cronin and William S. McIntire     

Papers to Appear in Forthcoming Issues

Purification of A Multicatalytic Protease Complex from Developing Winged Bean Seeds by Indirect Immuno-affinity ChromatographyRajamma Usha and Manoranjan SinghProduction of Reagents and Optimization of Methods for Studying Calmodulin-Binding ProteinsBettina Ulbricht and Thierry SoldatiExpression, Folding, and Characterization of Small Proteins with Increasing Disulfide Complexity by a pT7-7-Derived PhagemidFrancis C. Peterson, Patricia J. Anderson, Lawrence J. Berliner, and Charles L. BrooksDisulfide Bond Formation and Folding of Plant Peroxidases Expressed as Inclusion Body Protein inEscherichia coliThioredoxin Reductase Negative StrainsKaare Teilum, Lars Østergaard, and Karen G. WelinderOptimized Heterologous Expression of Glutathione Reductase from CyanobacteriumAnabaenaPCC 7120 and Characterization of the Recombinant ProteinFanyi Jiang and Bengt Mannervik     

The Conserved Modular Elements of the Acyl Carrier Proteins of Lipid Synthesis Are Only Partially Interchangeable

Prior work showed that expression of acyl carrier proteins (ACPs) of a diverse set of bacteria replaced the function of Escherichia coli ACP in lipid biosynthesis. However, the AcpAs of Lactococcus lactis and Enterococcus faecalis were inactive. Both failed to support growth of an E. coli acpP mutant strain. This defect seemed likely because of the helix II sequences of the two AcpAs, which differed markedly from those of the proteins that supported growth. To test this premise, chimeric ACPs were constructed in which L. lactis helix II replaced helix II of E. coli AcpP and vice versa. Expression of the AcpP protein L. lactis AcpA helix II allowed weak growth, whereas the L. lactis AcpA-derived protein that contained E. coli AcpP helix II failed to support growth of the E. coli mutant strain. Replacement of the L. lactis AcpA helix II residues in this protein showed that substitution of valine for the phenylalanine residue four residues downstream of the phosphopanthetheine-modified serine gave robust growth and allowed modification by the endogenous AcpS phosphopantetheinyl transferase (rather than the promiscuous Sfp transferase required to modify the L. lactis AcpA and the chimera of L. lactis AcpA helix II in AcpP). Further chimera constructs showed that the lack of function of the L. lactis AcpA-derived protein containing E. coli AcpP helix II was due to incompatibility of L. lactis AcpA helix I with the downstream elements of AcpP. Therefore, the origins of ACP incompatibility can reside in either helix I or in helix II.     

Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450

MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.     

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I)

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.     

Production and purification of an extracellularly produced K4 polysaccharide from Escherichia coli

Summary The production of the K4 polysaccharide was obtained for the first time extracellularly from a strain of Escherichia coli. The set up of the fermentation conditions led to the maximum fermentation yield, as extracellular K4, after 20 h. Purification and characterization of this K4 resulted in 200 mg/L of highly purified K4.     

Cloning of the Pachysolen tannophilus Xylulokinase Gene by Complementation in Escherichia coli

The gene coding for xylulokinase has been isolated from the yeast Pachysolen tannophilus by complementation of Escherichia coli xylulokinase (xylB) mutants. Through subcloning, the gene has been localized at one end of a 3.2-kilobase EcoRI-PstI fragment. Expression of the cloned gene was insensitive to glucose inhibition. Furthermore, the cloned gene did not cross-hybridize with E. coli and Saccharomyces cerevisiae xylulokinase genes.     

Overexpression of Neurospora crassa OR74A glutamate decarboxylase in Escherichia coli for efficient GABA production

This study investigated the effect of glutamate decarboxylase from Neurospora crassa OR74A on GABA production in Escherichia coli. GABA is one of the inhibitory neurotransmitters in the mammalian central nervous system, and can be used as a precursor of promising biopolymer Nylon 4. E. coli that overexpressed N. crassa glutamate decarboxylase was cultured at various pH levels and temperatures to determine optimum conditions for GABA production. When the recombinant E. coli strain was cultured at 30°C and pH 3, a final GABA concentration of 5.26 g/L was obtained from 10 g/L of monosodium glutamate (MSG), corresponding to a GABA yield of 86.23%.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.     

Dissection of 16S rRNA methyltransferase (KsgA) function in Escherichia coli

A 16S rRNA methyltransferase, KsgA, identified originally in Escherichia coli is highly conserved in all living cells, from bacteria to humans. KsgA orthologs in eukaryotes possess functions in addition to their rRNA methyltransferase activity. E. coli Era is an essential GTP-binding protein. We recently observed that KsgA functions as a multicopy suppressor for the cold-sensitive cell growth of an era mutant [Era(E200K)] strain (Q. Lu and M. Inouye, J. Bacteriol. 180:5243-5246, 1998). Here we observed that although KsgA(E43A), KsgA(G47A), and KsgA(E66A) mutations located in the S-adenosylmethionine-binding motifs severely reduced its methyltransferase activity, these mutations retained the ability to suppress the growth defect of the Era(E200K) strain at a low temperature. On the other hand, a KsgA(R248A) mutation at the C-terminal domain that does not affect the methyltransferase activity failed to suppress the growth defect. Surprisingly, E. coli cells overexpressing wild-type KsgA, but not KsgA(R248A), were found to be highly sensitive to acetate even at neutral pH. Such growth inhibition also was observed in the presence of other weak organic acids, such as propionate and benzoate. These chemicals are known to be highly toxic at acidic pH by lowering the intracellular pH. We found that KsgA-induced cells had increased sensitivity to extreme acid conditions (pH 3.0) compared to that of noninduced cells. These results suggest that E. coli KsgA, in addition to its methyltransferase activity, has another unidentified function that plays a role in the suppression of the cold-sensitive phenotype of the Era(E200K) strain and that the additional function may be involved in the acid shock response. We discuss a possible mechanism of the KsgA-induced acid-sensitive phenotype.     

An improved method to clone penicillin acylase genes: Cloning and expression in Escherichia coli of penicillin G acylase from Kluyvera citrophila

A simple and versatile procedure to clone penicillin acylase genes has been developed. It involves the construction of a plasmid library in a host presenting an amino acid auxotrophy. Recombinant clones carrying the acylase gene were selected on a minimal medium containing instead of the required amino acid its phenylacetyl derivative. Penicillin acylase genes from Escherichia coli ATCC 11105 and Kluyvera citrophila ATCC 21285 have been cloned in E. coli using this technique. The restriction map of the region containing the E. coli penicillin acylase gene was found to be similar to that described by H. Mayer et al. (in: Plasmids of Medical, Environmental and Commercial Importance (Timmis, K.M. and Paler, A., eds.), pp. 459–470, Elsevier, Amsterdam 1979). K. citrophila acylase gene was located within a 3.0 kb Hind III-PvuI fragment. Some differences were observed between the partial restriction maps of both genes. In addition, the production of those clones carrying the E. coli acylase was more sensitive to the growth temperature than that of the clones containing the K. citrophila gene. Bacteria harbouring plasmids containing the K. citrophila acylase sequence were able to produce about 30 fold more enzyme than the parental strain. A 60 000 dalton polypeptide corresponding to the K. citrophila acylase has been detected in a maxicell system. The industrial applications of the procedure are discussed.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc-α ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli

The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.     

Isolation of Antibiotics Turbomycin A and B from a Metagenomic Library of Soil Microbial DNA

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.