Characterization of auxin-binding protein 1 from tobacco: content, localization and auxin-binding activity

There is evidence that auxin-binding protein 1 (ABP1) is an auxin receptor on the plasma membrane. Maize (Zea mays L.) possesses a high level of auxin-binding activity due to ABP1, but no other plant source has been shown to possess such an activity. We have analyzed the ABP1 content of tobacco (Nicotiana tabacum L.) to examine whether or not the ABP1 content of maize is exceptionally high among plants. The ABP1 content of tobacco leaves was shown by quantitative immunoblot analysis to be between 0.7 and 1.2 μg ABP1 per gram of fresh leaf. This value is comparable to the reported value in maize shoots, indicating that ABP1 is present at a similar level in both monocot and dicot plants. The ABP1 content of tobacco leaves was increased up to 20-fold by expression of a recombinant ABP1 gene, and decreased to half of the original value by expression of the antisense gene. Although ABP1 was found mainly in the endoplasmic reticulum fraction, a secreted protein showing a molecular size and epitopes similar to intracellular ABP1 was also detected in the culture medium of tobacco leaf disks. The secretion of this protein was dependent on the expression level of the ABP1 gene. Received: 24 February 1999 / Accepted: 25 March 1999     

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts l- or d-glutamate to d- or l-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K _m and k_cat values of the overexpressed A. pyrophilus glutamate racemase for the racemization of l-glutamate to the d-form and the conversion of d-glutamate to the l-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06 s⁻¹ or 0.50 ± 0.07 mM and 0.25 ± 0.01 s⁻¹, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min. Received: September 11, 1998 / Accepted: January 12, 1999     

Cloning Vectors for Rice

We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc, respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning of full-length cDNA clones into our binary vectors.     

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80–90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young’s were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration.     

Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation,properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane

The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds. The fhuA gene encodes the outer membrane receptor protein. Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37° C large amounts of the proFhuA protein. Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis. Despite inhibition of export proFhuA synthesis continued.The precipitate formed was sedimented by centrifugation at 8,000xg. The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate. Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein. Binding of phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M. Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns. Addition of ferrichrome affected trypsin cleavage of both proteins in the same way. The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.Dedicated to Prof. G. Braunitzer on the occasion of his 60th birthday     

Overexpression of δ-Opioid Receptors in Recombinant Baculovirus-Infected Trichoplusia ni"High 5" Insect Cells

Abstract: "High 5" cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse δ-opioid receptor. The maximal binding capacity for the narcotic antagonist [³H]naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [³H][ d -penicillamine², d -penicillamine⁵]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at δ-receptors of NG108-15 hybrid cells and of High 5 and Sf9 insect cells. However, in insect cells the opioid was more than 100-fold less effective in competing with [³H]naltrindole as compared with the mammalian cells. This decline in potency was counteracted in a dose-dependent manner by exposure of High 5 membranes to the exogenous G protein G_o, which increased the binding capacity for DPDPE. Functional studies revealed a dose-dependent inhibition (up to 30%) by opioids on forskolin-stimulated cyclic AMP synthesis, and this effect was potentiated by G_o. Quantification of Gα_o and Gα_i disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned δ-opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of Gα_o/mg of membrane protein, and a 20-fold higher concentration for Gα_i. The distinct G-protein expression in insect cells may be considered an advantage for studying functions of G protein-coupled receptors.     

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli

Abstract Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi . This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD . The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains ix histidyl residues in its N-terminus.     

过量表达拟南芥CAT提高烟草对气体甲醛的吸收和抗性

为提高植物对甲醛的吸收和耐受,利用拟南芥CAT的编码区构建植物过表达载体pk2-Prbc S-*T-CAT,在野生型(WT)烟草叶绿体中过量表达CAT产生2个表达量不同的转基因株系(C1、C3)。用10ppm、20ppm和40ppm气体甲醛处理WT和转基因烟草,分析过量表达CAT的转基因烟草对甲醛的吸收效率和耐受性。结果表明,转基因烟草对气体甲醛的吸收量明显高于WT,且随着处理浓度的升高,转基因株系叶片的可溶性总糖、总蛋白质和总叶绿素含量都显著高于WT;转基因株系叶片氧化损伤指标H202、丙二醛(MDA)和羰基化蛋白(PC)的含量都显著低于野生型。转基因烟草(C1)的PM H+-ATPase和氢泵活性在40ppm处理后为WT的3倍和2.5倍,C1的气孔导度为WT的2.67倍。这些结果表明,在烟草中过量表达拟南芥CAT能降低甲醛进入烟草细胞引起的氧化损伤,提高烟草对甲醛的脱毒能力,增强烟草对甲醛的吸收和抗性。揭示气体甲醛胁迫时质膜H+-ATPase和氢泵活性影响叶片气孔的导度,这可能是转基因烟草甲醛吸收效率提高的原因之一。     

Bacterial expression of a eukaryotic membrane protein in fusion to various Mistic orthologs

Mistic, a bacterial membrane-associating protein family, uniquely found in Bacillus species. It enhances expression of eukaryotic membrane proteins at the bacterial membrane. Mistic from B. subtilis (M110), expresses at the Escherichia coli membrane, however its shorter orthologs have been recently shown to be mainly cytoplasmic with varying membrane affinities. Based on that, we hypothesized that the expression level of membrane proteins fused to Mistic is correlated with the degree of membrane association of the particular Mistic protein. We compared expression levels by various Mistic proteins as fusion partners for the Aplysia californica Kv1.1 (aKv1.1) channel as a cargo membrane protein. Mistic from B. atrophaeus (M4), which has the highest membrane association among the shorter orthologs, enhanced expression of the transmembrane domain of aKv1.1 to the highest extent. In contrast, M1, which consists of the 84 C-terminal amino acids of M110 is the most soluble protein and showed the least capacity to express the channel. A chimeric Mistic, constructed with the first α-helix (H1) of M110 N-terminally fused to M4, did not increase the level of expression of aKv1.1 beyond those of either the M110 or the M4 fusions. The channel fused to M110, M4 or the aforementioned H1-M4 chimera, expresses in the highest quantity and quality among Mistic proteins, providing suitable sample for structural studies. Our data support the concept that expression levels of ‘Misticated’ membrane proteins are related to the independent chaperoning character of Mistic via direct membrane association, rather than related to specific sequence-dependent interaction with the E. coli translocon machinery.