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

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     

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     

Purification and characterization of recombinantStreptomyces clavuligerus isopenicillin N synthase produced inEscherichia coli

Recombinant isopenicillin N synthase fromStreptomyces clavuligerus was produced in the form of inactive inclusion bodies inEscherichia coli. These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions. Optimization of refolding conditions to recover active isopenicillin N synthase indicated that a dialysis procedure carried out at a protein concentration of about 1.0 mg ml^–1 gave maximal recovery of active isopenicillin N synthase. Solubilized isopenicillin N synthase of more than 95% purity was obtained by passing this material through a DEAE-Trisacryl ion exchange column. Expression studies conducted at different temperatures indicated that isopenicillin N synthase was produced predominantly in a soluble, active form when expression was conducted at 20°C, and accounted for about 20% of the total soluble protein. This high-level production facilitated the purification of soluble isopenicillin N synthase to near homogeneity in four steps. Characterization of the purified soluble and solubilized isopenicillin N synthase revealed that they are very similar.     

M-like,immunoglobulin-binding protein ofStreptococcus pyogenes type M15

An M-like protein fromStreptococcus pyogenes type M15 strain EF1949 (EMML15) was cloned inEscherichia coli and sequenced. Recombinant EMML15 protein revealed a unique binding pattern for human IgG subclasses not described previously. Comparative analysis of the EMML15 amino acid sequence with those of other M-like proteins of opacity factor positive (OF⁺) serotypes and protein H, an IgG receptor from OF^– serotype M1, showed that IgG-binding proteins with common binding of IgG3 were closely related and distinct from streptococcal IgG receptors not binding IgG3. Thus, the Ig-binding proteins fromS. pyogenes were subdivided into two main categories according to binding pattern, protein structure, and gene location.     

4 Steps toward Inquiry Centered Teaching

Stidworthy, John. Life Begins. The Day of the Dinosaurs. Mighty Mammals of the Past. When Humans Began. Morristown, N.J.: Silver Burdett Co., 1986. Each 37 pp. $6.75 softcover Reveiwed by Donald J. Nash

McKay, David W., and Bruce G. Smith Space Science Projects for Young Scientists (Projects for Young Scientists series). New York: Franklin Watts, 1986. 127 pp. $ 10.90 hardcover (school and library binding) Reveiwed by Robert G. Hoehn

Griffin, Robert D. The Biology Coloring Book. Diamand, M. C., et al. The Human Brain Coloring Book. New York: Harper &; Row, 1986. Ill pp. (Biology) and 281 pp. (Brain) $9.95 paper

Kallenbach, Ernst. The Light Microscope: Principles and Practice for Biologists. Springfield, Il.: Charles C. Thomas, 1986. 56 pp. $14.25 softcover. Reveiwed by Robert E. Knowlton     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

Enzyme synthesis ofl-tryptophan

Enzyme synthesis of tryptophan from indole, pyruvate and ammonium salts was studied usingEscherichia coli cells exhibiting a significant tryptophanase activity. In addition to the effect of cultivation medium composition and cultivation conditions, factors affecting the course of the conversion were investigated. Production of 32.4 g/L ofl-tryptophan was reached after 48 h under optimal conditions.     

Computer game for protein folding

Computer game for protein folding Recombinant protein folding and misfolding in Escherichia coli Recent biotech patents on protein folding Protein location in the cell causes cancer Chemical engineer reveals the secret ingredient of the perfect sandwich Chemogenomics and open access to large-scale drug discovery data US drug patent expirations for August 2008     

ATP- and polyphosphate-dependent bacterial NAD+ kinases

Measurable levels of activity of NAD⁺ kinases of actinomycetesMicrococcus luteus andCoryne-bacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacteriumEscherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD⁺ kinase inC. ammoniagenes andE. coli; four forms were found inM. luteus. All isoforms of this enzyme found inC. ammoniagenes andM. luteus displayed NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate,M. luteus is the most promising NADP producer organism.     

Alcohol production from glucose and xylose usingEscherichia coli containingZymomonas mobilis genes

Summary AnEscherichia coli strain containing a recombinant plasmid encoding the pyruvate decarboxylase and alcohol dehydrogenase genes fromZymomonas mobilis metabolized glucose and xylose to near theoretical yields of ethanol. Enzyme activity measurements indicate high expression levels of both plasmid-encodedZymomonas proteins in the recombinantE. coli. The expression inE. coli is under the control of a promoter in theZymomonas sequence upstream of the pyruvate decarboxylase gene. The maximum ethanol level, using 4% glucose as substrate, was 1.8% (w/v) in anaerobic conditions. In aerobic conditions the natural repression ofE. coli alcohol dehydrogenase results in less ethanol production from clones expressing onlyZymomonas pyruvate decarboxylase.     

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently inEscherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved inde novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed inE. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced inE. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.     

The membrane binding C-terminus of protein A fromStaphylococcus aureus affects its cellular localization and causes structural deformation when expressed inEscherichia coli

Protein A fromStaphylococcus aureus is a powerful diagnostic reagent and has several uses in human disease therapy. Expression in non-pathogenicEscherichia coli containing recombinant plasmids coding for this protein has increased its availability, but can reduce the stability of the plasmid-bearing host. By employing immune electron microscopy, we have determined thatE. coli containing stable plasmids coding for a truncated version of protein A, without the membrane binding site, secrete this protein through the cytoplasmic membrane and into the periplasmic space, where it accumulates.E. coli containing unstable plasmids, however, which code for the complete protein including the membrane-binding site, target the protein into the cytoplasmic membrane. This accumulation of protein A in theE. coli cytoplasmic membrane inhibits the formation of septa between dividing cells and results in aberrant elongated, multi-chromosomal forms.     

rRNA topography in ribosome. IV. The accessibility of the 5'-end region of 16S rRNA

The accessibility of the 5'-end region of 16S rRNA (A₈GAGUUUG₁₅) inEscherichia coli ribosomes for complementary binding with the synthetic octanucleotide d(CAAACTCT) has been studied. Nonequilibrium gel-filtration was used to evaluate parameters of the binding of this oligonucleotide with free 16S rRNA, ribosomal subunits, and 70S ribosomes. A simple approach is presented to calculate the apparent association constants and the number of binding sites based upon the data obtained under those conditions. Free 16S rRNA, 30S subunits, and 70S ribosomes were found to form rather stable complexes with the octanucleotide, the association constants being similar in all three cases. These data strongly suggest the surface location of the 16S rRNA 5'-end inE. coli ribosomes.     

A Rab-related small GTP binding protein is predominantly expressed in root nodules of<Emphasis Type="Italic"> Medicago sativa</Emphasis>

Rab-related small GTP-binding proteins are known to be involved in the regulation of the vesicular transport system in eucaryotic cells. In this paper we report the isolation of the cDNA clone MS- rab11f from Medicago sativa (alfalfa) root nodules using a combination of RT-PCR and SSCP analysis. MS- rab11f shows high homology to the Rab-related cDNA clone LJ- rab11f from Lotus japonicus root nodules. The MS-Rab11F protein expressed in Escherichia coli was found to bind GTP, confirming that the isolated cDNA indeed codes for a small GTP-binding protein. Expression analysis by RT-PCR demonstrated that MS- rab11f is preferentially expressed in root nodules of alfalfa. Using the cDNA-sequence of MS-rab11f, a peptide-specific antibody was generated. Western blot analysis with this antibody revealed that two Rab11F isoforms, designated MS-Rab11FA and MS-Rab11FB, are found in M. sativa root nodules.Communicated by A. Kondorosi     

Factors affecting the expression of foreign proteins inEscherichia coli

Summary A variety of factors affect the expression of foreign proteins inEscherichia coli. These include: promoter strength, efficiency of ribosome binding, stability of the foreign protein inE. coli, location of the foreign protein inE. coli, the codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number of the foreign gene. This paper contains a critical review of these factors with the idea that a detailed understanding of them is the key to the development of strategies for the efficient large-scale production of foreign proteins inE. coli.     

Molecular cloning of thehom—thrC—thrB cluster fromBacillus sp. ULM1: Expression of thethrC gene inEscherichia coli and corynebacteria,and evolutionary relationships of the threonine genes

A 6.5 kb DNA fragment containing the gene (thrC) encoding threonine synthase, the last enzyme of the threonine biosynthetic pathway, has been cloned from the DNA ofBacillus sp. ULM1 by complementation ofEscherichia coli andBrevibacterium lactofermentum thrC auxotrophs. Complementation studies showed that thethrB gene (encoding homoserine kinase) is found downstream from thethrC gene, and analysis of nucleotide sequences indicated that thehom gene (encoding homoserine dehydrogenase) is located upstream of thethrC gene. The organization of this cluster of genes is similar to theBacillus subtilis threonine operon (hom—thrC—thrB). An 1.9 kbBclI, fragment from theBacillus sp. ULM1 DNA insert that complementedthrC mutations both inE. coli and in corynebacteria was sequenced, and an ORF encoding a protein of 351 amino acids was found corresponding to a protein of 37462 Da. ThethrC gene showed a low G+C content (39.4%) and the encoded threonine synthase is very similar to theB. subtilis enzyme. Expression of the 1.9 kbBclI DNA fragment inE. coli minicells resulted in the formation of a 37 kDa protein. The upstream region of this gene shows promoter activity inE. coli but not in corynebacteria. A peptide sequence, including a lysine that is known to bind the pyridoxal phosphate cofactor, is conserved in all threonine synthase sequences and also in the threonine and serine dehydratase genes. Amino acid comparison of nine threonine synthases revealed evolutionary relationships between different groups of bacteria. Dedicated to Dr. J. Spížek on the occasion of his 60th birthday     

An improved method for large-scale purification of recombinant human glucagon

Glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence [Ishizakiet al. (1992),Appl. Microbiol. Biotechnol.36, 483–486]. The high-level expression of a protein inE. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawaet al. (1992),J. Protein Chem. 11, 517–525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon.     

The Status and the Role of Glutathione under Disturbed Ionic Balance and pH Homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSH_in + GSSG_in) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 g/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSH_in : GSSG_in) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSH_out : GSSG_out) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.