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Volume 133, Issue 1
Displaying 1-19 of 19 articles from this issue
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Takaaki Hirai, Kazuhiro Chida2003 Volume 133 Issue 1 Pages 1-7
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe ζ isotype of protein kinase C (PKCCζ) is a member of the atypical PKC subfamily and has been widely implicated in the regulation of cellular functions. Increasing evidence from studies using in vitro and in vivo systems points to PKCζ as a key regulator of critical intracellular signaling pathways induced by various extracellular stimuli. The major activation pathway of PKCζ depends on phosphatidylinositol (PI)-3, 4, 5-trisphosphate (PIP3), which is mainly produced by PI-3 kinase. 3'-PI-dependent protein kinase 1, which binds with high affinity to PIP3, phosphorylates and activates PKCζ. Many studies demonstrated the involvement of PKCζ in the mitogen-activated protein kinase cascade, transcriptional factor NFKB activation, ribosomal S6-protein kinase signaling, and cell polarity. An important molecular event in a cell is the association of PKCζ with other signaling molecules, as well as scaffold proteins, to form large complexes that regulate their pathways. The understanding of the mechanisms underlying PKCζ-mediated control of intracellular signaling is beginning to provide important insights into the roles of PKCζ in various cells.View full abstractDownload PDF (3539K) -
Atsushi Suzuki, Kazunori Akimoto, Shigeo Ohno2003 Volume 133 Issue 1 Pages 9-16
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPKCλ/l belongs to the third group of the PKC family, atypical PKC (aPKC), together with PKCζ based on its sequence divergence from conventional and novel PKCs observed not only in the N-terminal regulatory domain but also in the kinase domain. Although one of the most distinct features of aPKC is its single, unrepeated cysteinerich domain, recent studies have revealed that the N-terminal regulatory domain has additional aPKC-specific structural motifs involved in various protein-protein interactions, which are important for the regulation and the subcellular targeting of aPKC. The identification of aPKC-specific binding proteins has significantly facilitated our understanding of the activation mechanism as well as the physiological function of aPKC at the molecular level. In particular, the finding that the mammalian homologs of the Caenorhabditis elegans proteins, PAR-3 and PAR-6, bind aPKC unexpectedly opens a new avenue for exploring a thus far completely unrecognized critical function of aPKC, that is, as a component of an evolutionarily conserved cell polarity machinery. Together with the great progress in the genome project as well as in the genetic analysis of model organisms, these advances are leading us into the new era of aPKC study in which functional divergence between PKCλ/l and ζ can be discussed in elaborately.View full abstractDownload PDF (3288K) -
Hideyuki Mukai2003 Volume 133 Issue 1 Pages 17-27
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPKN is a serine/threonine protein kinase that has a catalytic domain homologous to protein kinase C (PKC) family members and a unique regulatory region containing antiparallel coiled-coil (ACC) domains. PKN is the first identified serine/threonine protein kinase that can bind to and be activated by a small GTPase Rho, and it can also be activated by fatty acids such as arachidonic acid in vitro. PKN is widely distributed in various organisms such as mammal, frog, fly, and starfish. There are at least three different isoforms of PKN (PKNα/PAK-1/PRK-l, PKNβ, and PRK2/PAK-2/ PKNγ) in mammals, each of which shows different enzymological properties, tissue distribution, and varied functions.View full abstractDownload PDF (2815K) -
Daisuke Irikura, Takashi Kumasaka, Masaki Yamamoto, Hideo Ago, Masashi ...2003 Volume 133 Issue 1 Pages 29-32
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSLipocalin-type prostaglandin D synthase is the key enzyme for the production of prostaglandin D2, a potent endogenous somnogen, in the brain. We cloned, produced, and crystallized the native enzyme and selenomethionyl Cys65Ala mutants of the recombinant mouse protein by the hanging drop vapor-diffusion method with both malonate and citrate as precipitants. The native crystals obtained with malonate belong to orthorhombic space group P212121 with lattice constants a=46.2, b=66.8, and c=105.3 Å. The selenomethionyl crystals obtained with citrate belong to orthorhombic space group C2221 with lattice constants a=45.5, b=66.8, and c=104.5 Å. The native crystals diffracted beyond 2.1 Å resolution.View full abstractDownload PDF (1406K) -
Noriaki Arakawa, Motoki Igarashi, Takayuki Kazuoka, Tadao Oikawa, Kenj ...2003 Volume 133 Issue 1 Pages 33-42
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSD-Arginase activity was found in the cells of an isolate, Arthrobacter sp. KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized. Its molecular weight was estimated to be about 232, 000 by gel filtration, and that of the subunit was approximately 40, 000 by SDS-PAGE, suggesting that the enzyme is a homohexamer. The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine. The Vmax/Km values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 μmol/min/mg/mM, respectively. Accordingly, the enzyme is regarded as a kind of guanidinobutyrase [EC 3.5.3.7]. The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively. The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors. The enzyme contained about 1 g-atom of firmly bound Zn2+ per mol of subunit, and removal of the metal ions by incubation with 1, 10-phenanthroline resulted in loss of activity. The inactivated enzyme was reactivated markedly by incubation with either Zn2+ or Co2+, and slightly by incubation with Mn2+. The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (Mr: 37, 933). The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn2+-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).View full abstractDownload PDF (1364K) -
Shuhei Hashiguchi, Toshihiro Nakashima, Aya Nitani, Tomoki Yoshihara, ...2003 Volume 133 Issue 1 Pages 43-49
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe α-chain of FcεRI (FcεRIα) plays a critical role in the binding of IgE to FcεRI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human FcεRIα using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcRε27, FcRε51, and FcRε70) through two rounds of biopanning selection. The purified soluble scFv, FcRε5l, inhibited the binding of IgE to recombinant FcεRIα, although both FcRε27 and FcRε70 showed fine binding specificity to FcεRIα. Since FcRε51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcRε51 to FcεRIα was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to FcεRIα (Kd=0.5nM). With these characteristics, FcRε51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.View full abstractDownload PDF (970K) -
Takayuki Kazuoka, Yuki Masuda, Tadao Oikawa, Kenji Soda2003 Volume 133 Issue 1 Pages 51-58
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe found that a psychrophilic bacterium isolated from Antarctic seawater, Cytophaga sp. KUC-1, abundantly produces aspartase [EC4.3.1.1], and the enzyme was purified to homogeneity. The molecular weight of the enzyme was estimated to be 192, 000, and that of the subunit was determined to be 51, 000: the enzyme is a homotetramer. L-Aspartate was the exclusive substrate. The optimum pH in the absence and presence of magnesium ions was determined to be pH 7.5 and 8.5, respectively. The enzyme was activated cooperatively by the presence of L-aspartate and by magnesium ions at neutral and alkaline pHs. In the deamination reaction, the Km value for L-aspartate was 1.09mM at pH 7.0, and the S1/2 value was 2.13mM at pH 8.5. The Vmax value were 99.2U/mg at pH 7.0 and 326 U/mg at pH 8.5. In the amination reaction, the Km values for fumarate and ammonium were 0.797 and 25.2mM, respectively, and Vmax was 604U/mg. The optimum temperature of the enzyme was 55°C. The enzyme showed higher pH and thermal stabilities than that from mesophile: the enzyme was stable in the pH range of 4.5-10.5, and about 80% of its activity remained after incubation at 50°C for 60 min. The gene encoding the enzyme was cloned into Escherichia coli, and its nucleotides were sequenced. The gene consisted of an open reading frame of 1, 410-bp encoding a protein of 469 amino acid residues. The amino acid sequence of the enzyme showed a high degree of identity to those of other aspartases, although these enzymes show different thermostabilities.View full abstractDownload PDF (1022K) -
Wei-Gang Hu, Azhar Z. Alvi, Damon Chau, Jeffrey E. Coles, R. Elaine Fu ...2003 Volume 133 Issue 1 Pages 59-66
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMurine monoclonal antibody 1A4A1 has been shown to recognize a conserved neutralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus. It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy. In order to minimize the immunogenicity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed. It encoded a human IgG1 heavy chain constant region and a single-chain fragment variable antibody of 1A4A1. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recognition by protein G and human complement C1q binding. On non-reducing and reducing gel electrophoresis analysis of proteolytic fragments of the recombinant antbody, disulfide bond formation was found in the hinge region of the antibody. From these data, it was concluded that the recombinant antibody was capable of antigen recognition, and retained several functional activities. This work forms the basis for characterization of the recombinant antibody as to efficacy in vivo.View full abstractDownload PDF (4158K) -
Michiyo Nomura, Shigeru Nakamori, Hiroshi Takagi2003 Volume 133 Issue 1 Pages 67-74
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe recently found that budding yeast Saccharomyces cerevisiae Σ1278b, but not genome project strain S288C, has a gene conferring resistance to L-azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline. Also, the gene, designated as MPR1, encodes a novel acetyltransferase that detoxifies AZC via acetylation. We now report the results of subsequent work. On a homology search with MPR1, we detected a gene in fission yeast Schizosaccharomyces pombe. This gene, designated as pprl+ (pombe MPR1), is responsible for the AZC-resistance of S. pombe as judged from the results of gene disruption and overexpression experiments. Escherichia coli cells expressing ppr1+, like ones expressing MPR1, were resistant to AZC and produced an AZC acetyltransferase. We further found that the enzymes encoded by MPR1 and ppr1+ were homodimers, and catalyzed the acetylation of AZC but not any other L-proline-related compounds. Ppr1p was more thermostable than Mpr1p, although Ppr1p had a lower optimum temperature than Mprlp. The higher AZC acetylation activity of Mpr1p, in comparison to that of Ppr1p, was attributed to the larger kcat/Km value for acetyl-CoA of Mpr1p than that of Ppr1p.View full abstractDownload PDF (2259K) -
Min Yao, Akiko Ohsawa, Shingo Kikukawa, Isao Tanaka, Makoto Kimura2003 Volume 133 Issue 1 Pages 75-81
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSEukaryotic initiation factor 5A (eIF-5A) is ubiquitous in eukaryotes and archaebacteria and is essential for cell proliferation and survival. The crystal structure of the eIF-5A homologue (PhoIF-5A) from a hyperthermophilic archaebacterium Pyrococcus horihoshii OT3 was determined at 2.0 Å resolution by the molecular replacement method. PhoIF-5A is predominantly composed of β-strands comprising two distinct folding domains, an N-domain (residues 1-69) and a C-domain (residues 72-138), connected by a short linker peptide (residues 70-71). The N-domain has an SH3-like barrel, while the C-domain folds in an (oligonucleotide/oligosaccharide binding) OB fold. Comparison of the structure of PhoIF-5A with those of archaeal homologues from Methanococcus jannaschii and Pyrobaculum aerophilum showed that the N-domains could be superimposed with root mean square deviation (rmsd) values of 0.679 and 0.624 Å, while the C-domains gave higher values of 1.824 and 1.329 Å, respectively. Several lines of evidence suggest that eIF-5A functions as a biomodular protein capable of interacting with protein and nucleic acid. The surface representation of electrostatic potential shows that PhoIF-5A has a concave surface with positively charged residues between the N- and C-domains. In addition, a flexible long hairpin loop, L1 (residues 33-41), with a hypusine modification site is positively charged, protruding from the N-domain. In contrast, the opposite side of the concave surface at the C-domain is mostly negatively charged. These findings led to the speculation that the concave surface and loop L1 at the N-domain may be involved in RNA binding, while the opposite side of the concave surface in the C-domain may be involved in protein interaction.View full abstractDownload PDF (2799K) -
Takashi Kawasaki, Yoshimitsu Hamano, Tomohisa Kuzuyama, Nobuya Itoh, H ...2003 Volume 133 Issue 1 Pages 83-91
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPrenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) Biosci. Biotechnol. Biochem. 65, 1627-1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the -50th, -8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity.View full abstractDownload PDF (2013K) -
A. A. Moosavi-Movahedi, J. Chamani, Y. Goto, G. H. Hakimelahi2003 Volume 133 Issue 1 Pages 93-102
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe molten globule state of cytochrome c is the major intermediate of protein folding. Elucidation of the thermodynamic mechanism of conformational stability of the molten globule state would enhance our understanding of protein folding. The formation of the molten globule state of cytochrome c was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS, at low concentrations. The refolding states of the protein were monitored by spectroscopic techniques including circular dichroism (CD), visible absorbance and fluorescence. The effect of n-alkyl sulfates on the structure of acid-unfolded horse cytochrome c at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of the molten globule state. The addition of n-alkyl sulfates to the unfolded state of cytochrome c appears to support the stabilized form of the molten globule. The m-values of the refolded state of cytochrome c by SOS, SDeS, SDS, and STS showed substantial variation. The enhancement of m-values as the stability criterion of the molten globule state corresponded with increasing chain length of the cited n-alkyl sulfates. The compaction of the molten globule state induced by SDS, as a prototype for other n-alkyl sulfates, relative to the unfolded state of cytochrome c was confirmed by Stokes radius and thermal transition point (Tm) measured by microviscometry and differential scanning calorimetry (DSC), respectively. Thus, hydrophobic interactions play an important role in stabilizing the molten globule state.View full abstractDownload PDF (1199K) -
Jaesun Chun, Taegun Kwon, Dae Joong Kim, Ingun Park, Guhung Chung, Eun ...2003 Volume 133 Issue 1 Pages 103-108
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe mitogen-activated protein kinase kinase kinase 3 (MEKK3) is a member of the MAP kinase family whose cellular activity is elevated in response to growth factors, oxidative stress, and hyperosmolar conditions. MEKK3 regulates MKK3 and MKK5/6/7. MEKK3 is involved distinctively in the signal pathway for blocking cell proliferation and cell cycle progression, contradictory to the biological responses commonly associated with other members of MEKKs. Based information concerning the substrate specificity of serum- and glucocorticoid-induced kinase 1 (SGK1), R-x-R-x-x-(S/T)-φ, where φ indicates a hydrophobic amino acid, two putative phosphorylation sites (Ser166 and Ser337) were found in MEKK3. It was shown that the recombinant MEKK3 protein and fluorescein-labeled MEKK3 peptides (FITC-159epRsRhlSVi168 and FITC-330dpRgRlpSAd339) are phosphorylated by SGK1 in vitro. It was also observed that the intrinsic kinase activity of MEKK3 on Ser189 of MKK3 (equivalent to Ser207 of MKK6) decreased along with phosphorylation of Ser166 and Ser337 in MEKK3 in vitro and in vivo. Therefore, it is suggested that SGK1 inhibits MEKK3-MKK3/6 signal transduction by phosphorylation of MEKK3.View full abstractDownload PDF (4230K) -
Takaho Endo, Atsuo Sasaki, Mayu Minoguchi, Akiko Joo, Akihiko Yoshimur ...2003 Volume 133 Issue 1 Pages 109-113
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSProlactin (PRL) interacts with a single-chain prolactin-specific receptor of the cytokine receptor superfamily. PRL triggers the activation of JAK2 kinase, which phosphorylates the PRL receptor itself, and of STAT5, a member of the family of signal transducers and activators of transcription (STAT). We have shown that the STAT5-dependent immediate early gene, CIS1 (Cytokine-Inducible SH2 domain-containing protein-1), suppresses PRL-induced STAT5 activation in vitro as well as in transgenic mice. To facilitate the study of the interactions between CIS1 and the PRL receptor, we have developed the yeast tri-hybrid system, a modification of the yeast two-hybrid system. We expressed CIS1 fused to the DNA-binding domain and PRL receptor cytoplasmic domain fused to the transcription activation domain in the presence or absence of the tyrosine kinase domain of JAK2 in yeast. CIS1 bound to the PRL receptor cytoplasmic domain in a JAK2-dependent manner. Moreover, we determined that the phosphorylated Y532 of the murine PRL receptor is the binding site for CIS1. Interestingly, Y532 has been shown to be unnecessary for STAT5 activation, although CIS1 overexpression suppressed PRL-induced STAT5 activation. These data suggest that the suppression of STAT5 activation by CIS1 is not due to a simple competition with STAT5 but rather to a modification of the receptor by CIS1 binding.View full abstractDownload PDF (1756K) -
Jingfang Zhao, Toshimitu Onduka, Jun-ya Kinoshita, Masanori Honsho, To ...2003 Volume 133 Issue 1 Pages 115-121
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSubfractionation studies showed that cytochrome b5 (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxyterminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.View full abstractDownload PDF (3560K) -
Shunsuke Kawamura, Tamo Fukamizo, Tomohiro Araki, Takao Torikata2003 Volume 133 Issue 1 Pages 123-131
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTo investigate the structure-function relationships of goose-type lysozyme, a gene coding for ostrich egg-white lysozyme (OEL) was designed based on the published amino acid sequence and constructed by assembling 32 chemically synthesized oligonucleotides. To obtain the recombinant OEL (rOEL), the synthetic gene was fused to the α-factor signal peptide in the expression vector pPIC9K and expressed in the methylotrophic yeast Pichia pastoris. The secreted protein from the transformed yeast was found to be processed at three different sites, including the correct site. The correctly processed rOEL was purified to homogeneity and shown to be indistinguishable from the authentic form in terms of circular dichroism (CD) spectrum and enzyme activity. Furthermore, the time-course of the reaction catalyzed by OEL was studied using (G1cNAc)n (n=5 and 6) as the substrate and compared to that of goose egg-white lysozyme (GEL) [Honda and Fukamizo (1998) Biochim. Biophys. Acta 1388, 53-65]. OEL hydrolyzed (GIcNAc)6 in an endo-splitting manner producing mainly (G1cNAc)2, (G1cNAc)3, and (G1cNAc)4, and cleavage to (GlcNAc)3+(GlcNAc)3 predominated over that to (GlcNAc)2+(GlcNAc)4. This indicates that OEL hydrolyzes preferentially the third glycosidic linkage from the nonreducing end of (GlcNAc)6 as in the case of GEL. The cleavage pattern seen for (GlcNAc)5 was similar to that seen for (GlcNAc)6. Theoretical analysis of the reaction time-course for OEL revealed that the binding free energy values for subsites B, E, and G were different between OEL and GEL, although these lysozymes were estimated to have the same type of subsite structure.View full abstractDownload PDF (1023K) -
2Jun Hasegawa, Masaki Endou, Takashi Narita, Tomoko Yamada, Yuki Yamagu ...2003 Volume 133 Issue 1 Pages 133-138
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe molecular dissection of transcription mechanisms is greatly facilitated by constructing and manipulating defined transcription systems in vitro. This approach requires highly purified transcription factors. A major enzyme participating in the transcription reaction is RNA polymerase II (RNAPII), which is composed of at least 12 subunits (RPB1-12). Due to its complex structure, it is difficult to prepare highly pure RNAPII by the conventional purification procedure. We transfected HeLa cells with a plasmid expressing RPB3 with a double FLAG-histidine tag on its amino-terminus. A high yielding clone was isolated and its extracts were subjected to immunoaffinity purification and then Co2+ affinity chromatography. This resulted in a preparation of RNAPII complexes that consisted of all the core subunits, including the double-tagged RPB3 protein. Transcription reactions with oligo (dC)-tailed templates and transcription assays involving general transcription factors revealed that the double-tagged RNAPII complexes are active and functional in basal and activated transcription. Our method is superior to the conventionally used purification procedure in that the final preparation is markedly more pure (92% versus 40%), and the procedures are much less time-consuming. Thus, this two-step affinity purification method is an uncomplicated and effective method by which active and functional RNAPII can be prepared.View full abstractDownload PDF (2755K) -
Kazuma Takase, Seiichi Taguchi, Yoshiharu Doi2003 Volume 133 Issue 1 Pages 139-145
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSType II synthase (PhaClps) for polyhydroxyalkanoate (PHA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagen-esis in order to improve the function of PhaClps in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Eseherichia coli. Based on our established in vivo assay system, two positions (Ser325 and G1n481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.View full abstractDownload PDF (899K) -
Juming Yao, Tetsuo Asakura2003 Volume 133 Issue 1 Pages 147-154
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSGenetic engineering strategies were applied to synthesize silk-like materials, [(GVPGV)2GG(GAGAGS)3AS]n. The primary structure of these materials represents the repetitive crystalline region of Bombyx mori silk fibroins incorporated with an elastic motif selected from animal elastin. The oligonucleotides were designed to encode the desired recombinant proteins and then expressed in the Escherichi coli system. The expression and purification conditions for the production of the recombinant proteins were optimized. 13C CP/MAS NMR was used for structural characterization in the solid state, where the isotope labeling was performed using a modified M9 medium. The secondary structures of these materials are primarily governed by the designated amino acid sequence, where the B. mori silk fibroin block, (GAGAGS)3, tends to form the crystalline region, which is interrupted by the flexible (GVPGV)2 block. The CD data suggested that the structure of these materials was length-dependent in the solution state, i. e., a higher molecule weight leads to a higher ordered structure.View full abstractDownload PDF (2993K)
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