Structure and expression of a hybrid proline-rich protein gene in the solanaceous species,Solanum brevidens, Solanum tuberosum, andLycopersicum esculentum

The full-length cDNA of a previously identified Solanum brevidens gene was isolated and characterised. DNA sequence analysis revealed an open reading frame that encodes a hybrid proline-rich cell wall protein of 407 amino acids. The putative protein was designated SbrPRP. The SbrPRP harbours three parts, an N-terminal signal peptide followed by a repetitive proline-rich domain and a cysteine-rich C-terminus resembling non-specific lipid-transfer proteins. The repetitive proline-rich domain contains two repeated motifs, PPHVKPPSTPK and PTPPIVSPP extended with TPKYP and TPKPPS motifs, respectively, at their N- or C-terminal. The SbrPRP gene of the non-tuberising Solanum species, Solanum brevidens, possesses highly homologous counterparts in the tuberising species, Solanum tuberosum (StPRP) and in the related species, Lycopersicum esculentum (TFM7). All three genes are present in single- or low copy number in the corresponding genome. Organ-specific expression of the genes, however, is different in the three solanaceous species.     

Structure,function, and biogenesis of SecY,an integral membrane protein involved in protein export

TheE. coli secY (prlA) gene, located in the operator-distal part of thespec ribosomal protein operon, codes for an integral membrane protein, SecY. The phenotypes of temperature-sensitive and cold-sensitive mutations insecY suggest that the SecY protein plays an essential rolein vivo to facilitate protein translocation, whereas theprlA mutations in this gene suggest that SecY may interact with the signal sequence of translocating polypeptides. SecY contains most probably six cytoplasmic and five periplasmic domains, as well as 10 transmembrane segments. Such membrane-embedded structure may confer the SecY protein a translocator function, in which it provides a proteinaceous pathway for passage of secreted as well as membrane proteins. Results obtained byin vitro analyses of the translocation reactions, as well as some new phenotypes of thesecY mutants, are consistent with this notion. Possible interaction of SecY with other secretion and chaperone-like factors is also discussed.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.     

AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development

The glycine-rich protein AtGRP2 is one of the four members of the cold-shock domain (CSD) protein family in Arabidopsis. It is characterized by the presence of a nucleic acid-binding CSD domain, two glycine-rich domains and two CCHC zinc-fingers present in nucleic acid-binding proteins. In an attempt to further understand the role of CSD/GRP proteins in plants, we have proceeded to the functional characterization of the AtGRP2 gene. Here, we demonstrate that AtGRP2 is a nucleo-cytoplasmic protein involved in Arabidopsis development with a possible function in cold-response. Expression analysis revealed that the AtGRP2 gene is active in meristematic tissues, being modulated during flower development. Down-regulation of AtGRP2 gene, using gene-silencing techniques resulted in early flowering, altered stamen number and affected seed development. A possible role of AtGRP2 as an RNA chaperone is discussed.     

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.     

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

Properties of the bubble protein, a defensin and an abundant component of a fungal exudate

Colonies of the ascomycete fungus Penicillium brevicompactum Dierckx produce bright yellow-green fluorescent exudate bubbles on its surface when grown on standard plant cell culture medium. According to SDS-PAGE analysis, the exudate is enriched in one protein, named bubble protein (BP). Detailed characteristics of BP are described, and also its corresponding genomic promoter and terminator sequences that flank sequences encoding signal peptide and a precursor sequence upstream of that of the mature protein. Following on previous work, the protein is now biochemically characterized. BP, the structure of which mainly consists of beta sheets, has four very stable disulfide bridges that resist standard procedures for reduction. With such traits, BP can now be categorized as a new member of the ever growing class of defensins. Indeed, the protein revealed anti-fungal effects as it inhibits growth of the yeast Saccharomyces cerevisiae in a dose-dependent manner. Structural classification places BP into the group of proteins with a knottin fold, founding the BP superfamily. Based on genomic alignments that revealed very high homology to four proteins of related fungi, a 3D structure prediction of the corresponding proteins was made. In addition, it was discovered that the closely related fungus Penicillium chrysogenum encodes a BP homolog – in addition to its PAF protein, which also is similar to BP – further suggesting that fungi may possess more than one defensin.     

Responses of Ribulose-1,5-Bisphosphate Carboxylase,Protein Content,and Stomatal Conductance to Water Deficit in Maize,Tomato, and Bean

We compared responses of maize, tomato, and bean plants to water stress. Maize reached a severe water deficit (leaf water potential –1.90 MPa) in a longer period of time as compared with tomato and bean plants. Maize stomatal conductance (g _s) decreased at mild water deficit. g _s of tomato and bean decreased gradually and did not reach values as low as in maize. The protein content was maintained in maize and decreased at low water potential (_w); in tomato it fluctuated and also decreased at low _w; in bean it gradually decreased. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity remained high at mild and moderate stress in maize and tomato plants; in bean it remained high only at mild stress.     

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.     

Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae

In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with α-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The soluble expression of the human renin binding protein using fusion partners: A comparison of ubiquitin,thioredoxin, maltose binding protein and NusA

human renin binding protein (hRnBp), showingN-acetylglucosamine-2-epimerase activity, was over-expressed inE. coli, but was mainly present as an inclusion body. To improve its solubility and activity, ubiquitin (Ub), thioredoxin (Trx), maltose binding protein (MBP) and NusA, were used as fusion partners. The comparative solubilities of the fusion proteins were, from most to least soluble: NusA, MBP, Trx, Ub. Only the MBP fusion did not significantly reduce the activity of hRnBp, but enhanced the stability. The Origami (DE3), permitting a more oxidative environment for the cytoplasm inE. coli, helped to increase its functional activity.     

Mass-spectrometric Identification of Binding Proteins of <Emphasis Type="BoldItalic">M</Emphasis><Subscript>r</Subscript> 25,000 Protein,a Part of Vitellogenin B1, Detected in Particulate Fraction of <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.     

UV-inducible proteins and UV-induced cross-protection against acid,ethanol, H2O2 or heat treatments in Lactococcus lactis subsp. lactis

The relationship between UV-irradiation-induced tolerance to different environmental stresses and change in protein synthesis was examined in Lactococcus lactis subsp lactis IL1403. The results showed that preirradiation of cultures of L. lactis subsp. lactis with UV_254nm light led to increased tolerance of usually lethal challenges to acid (pH 4.0), ethanol (20%, v/v), H₂O₂ (15 mM), or heat (52° C). This suggests that there is an overlapping regulation between the UV-induced pathway and the other stress responses. Whole-cell protein extracts from UV-treated (100 J/m²) and untreated cultures were compared using two-dimensional polyacrylamide gel electrophoresis. At least 14 polypeptides were induced in response to damage after UV irradiation, which indicated an SOS-like response in this species. The RecA protein, however, seemed not to be significantly induced in Lactococcus lactis subsp. lactis IL1403. Some of the UV-induced polypeptides overlaped with stress proteins induced by the other treatments.     

A stress-induced,developmentally regulated,highly polymorphic protein family in Pisum sativum L.

The expression of members of two closely related abscisic acid (ABA)-responsive pea protein families, ABR17 and ABR18 (ABA-responsive 17200-M_r and 18100-M_r, respectively), is developmentally, tissueand stress-specifically regulated. Two-dimensional polyacrylamide gel electrophoresis revealed a number of ABR polypeptides on fluorographs of immunoprecipitated translation products of mRNAs, depending on the tissue, stage of development or type of stress. High endogenous ABA, or added ABA, enhanced the accumulation of translatable mRNA for specific ABR members under certain conditions, but high endogenous ABA was not a pre-requisite for accumulation of translatable ABR mRNA. The accumulation of ABR polypeptides was examined by Western blot analysis of acetate-buffer-extracted proteins. In fully expanded, young unstressed leaves, the ABR17 polypeptides (ABR18 polypeptides not detectable) accumulated to markedly higher levels in the epidermis than in the mesophyll. Dehydration stress caused an increased (ABR17) and detectable (ABR18) polypeptide accumulation which occurred predominantly in the epidermis. Detached leaves were used further to characterise factors affecting ABR polypeptide accumulation. An enhanced (ABR17) and detectable (ABR18) polypeptide accumulation occurred in the presence of ABA (10^–4 M) but ABR18-polypeptide accumulation required light. The accumulation of both ABR polypeptides was stimulated in the presence of metabolisable and non-metabolisable carbohydrate sources but not in water or glutamine, indicating an osmotic rather than metabolic response. This carbohydrate-stimulated accumulation was markedly enhanced by light but unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis, indicating other photoreceptive processes besides photosynthesis were involved. The function of the ABR proteins remains unknown but their accumulation in aging tissues indicates a role in senescence. The results clearly demonstrate highly complex interactions between different environmental and developmental signals leading to the expression of these stressrelated proteins. In light of these results, the induction of protein expression of the newly-termed intracellular pathogenesis-related proteins, to which the ABR proteins are closely related, is discussed.Abbreviations ABA (±)cis, trans-abscisic acid - ABR17 M_r17200 ABA-responsive protein - ABR18 M_r-18100 ABA-responsive protein - 2-D two-dimensional - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FW fresh weight - IgG immunoglobulin G - M_r apparent molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Isolation, Crystallization, and Investigation of Ribosomal Protein S8 Complexed with Specific Fragments of rRNA of Bacterial or Archaeal Origin

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8–RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.     

Localization of a protein,immunologically similar to a sucrose-binding protein from developing soybean cotyledons,on the plasma membrane of sieve-tube members of spinach leaves

Immunocytochemical studies using antibodies raised against a 62-kDa membrane protein isolated from developing soybean (Glycine max (L.) Merr.) cotyledons were performed on leaf tissue of spinach (Spinacia oleracea L.). This 62-kDa protein was labeled by 6′-deoxy-6′-(4-azido-2-hydroxy)-benzamidosucrose (HABS), a photoaffinity sucrose analogue (K. G. Ripp et al., 1988, Plant Physiol.88, 1435–1445). Western-blot analysis of spinach plasma-membrane proteins indicated a cross-reactive polypeptide identical in molecular mass to that found in soybean. Indirect immunogold labeling of resin-embedded sections of fully expanded leaf tissue resulted in specific localization of colloidal gold on the sieve-tube plasma membrane. The label was uniform and, except for a few non-specific gold particles over the cell wall, all other cellular organelles and membrane systems were free of label. With the exception of occasional gold particles associated with the companion-cell plasma membrane, all other cell types of the leaf contained little or no label. Control sections treated with non-immune rabbit immunoglobulin-G were also essentially free of label. Immunogold labeling of young leaves, in which the phloem contained no mature sieve-tube members, were free of label for the 62-kDa protein. However, young leaf tissue in which mature or nearly mature sieve tubes could be identified, contained immunolabel associated with the sieve-tube plasma membranes. Similar results were obtained with mature leaf tissue of sugar beet (Beta vulgaris L.). The results of the immunocytochemical studies are consistent with the suggestion that the concentrating step in the phloem-loading process in this species may occur across the sieve-tube plasma membrane. This paper is dedicated to the memory of William A. Dungey