Expression of the Stress-Related Genes for Glutathione S-Transferase and Ascorbate Peroxidase in the Most-Glycinin-Deficient Soybean Cultivar Tousan205 during Seed Maturation

Global analysis of gene expression profiles in most-glycinin-deficient cultivar Tousan205, was performed by DNA microarray analysis. It was confirmed that Tousan205 lacks mRNA expression of three glycinin subunit precursor genes, G1 (A1aB1x), G2 (A2B1a), and G5 (A3B4), and lacks G4 (A5A4B3) protein. Most glycinin subunits were deficient in mature seeds of Tousan205. We compared the gene expression of Tousan205 with those of parent cultivar, Tamahomare, which was used for crossbreeding of Tousan205. As a result, Tousan205 exhibited higher expression of some seed maturation proteins, and stress-related genes such as glutathione S-transferase and ascorbate peroxidase. This result indicates the possibility that the decrease of main storage protein, glycinin causes stress in soybean.     

Signal sequence of preproglycinin affects production of the expressed protein in Escherichia coli

The effect of the signal peptide portion on the bacterial production of preproglycinin, a precursor of soybean storage protein, was examined. Nucleotide sequences corresponding to the signal peptide and the mature N-terminal region were deleted stepwise from the cDNA encoding the glycinin A1aB1b subunit precursor, and the deleted cDNAs were placed under the control of trc promoter in an expression vector pKK233-2. When the amounts of the protein products in Escherichia coli from each expression plasmid were determined, no accumulation of preproglycinin was observed from the plasmids with the full length or the five amino acids of the signal sequence. However, significant accumulation of the preproglycinin homologue proteins was noted from the plasmids retaining less than three amino acids of the signal sequence depending on the extent of deletion. N-terminal amino acid sequences of the products coincided with those predicted from the deleted cDNAs. The preproglycinin homologue proteins expressed from the mutant plasmids assembled into trimers of about 8S.     

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.     

Nucleotide sequence of cDNA of glicinin A3 subunits: variability of different species detected by comparative analysis

Several cDNA cloned which code for subfamily A3 subunits (A3B4 and A5A4B3) of soybean storage protein glycinin were analysed by means of restriction mapping and hybrid - selection. The comparison of A3B4 and A5A4B3 subunits cDNA sequences reveals the 90% homology. The nucleotide sequences obtained in the process of this work were compared with those reported about elsewhere, in order to study the interaspecific variability of homologous but not identical storage protein genes of subfamily A3 glycinin subunit. Nucleotide replacements were found to occur 6 times more frequently in A3B4 subunit gene, as compared to A5A4B3 subunit gene (48 replacements against 8).     

Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors

Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards.     

Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli.

The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed E1A and E1B. cDNAs containing the entire nucleotide sequence of the mature E1A 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from the Escherichia coli lac promoter. Upon transformation into appropriate E. coli hosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmid-encoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 E1A 42- to 50-kd and E1B 53- to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5'-proximal AUG.     

Novel approach to obtain biologically active recombinant heterodimeric proteins in Escherichia coli

The strategy described in this paper provides a novel approach for recombinant expression of heterodimeric proteins, and is especially suitable for the production of proteins whose characteristics lead to aggregation in E. coli expression systems. Pheromaxein, a steroid-binding protein isolated from boar saliva, is a heterodimeric protein consisting of 10x10(3) rel. mol. mass units (pheromaxein A) and 8x10(3) rel. mol. mass units (pheromaxein C) subunits. Expression of pheromaxein subunits in E. coli resulted in extensive insoluble aggregation. The difficulty faced in obtaining soluble recombinant pheromaxein subunits was clearly evident when native pheromaxein immediately formed aggregates when it was separated into its individual subunits. An increase in soluble pheromaxein expression in E. coli was obtained when the subunits were expressed as fusion proteins with thioredoxin. Pheromaxein genes were inserted separately into pET32a+ vectors at the NcoI site, resulting in thioredoxin, S.Tag and His.Tag coding regions being upstream of the inserted gene. Soluble pheromaxein A-thioredoxin (pheroA/trx) and pheromaxein C-thioredoxin (pheroC/trx) fusions were purified to homogeneity, using a laboratory scale S-protein agarose affinity column. Cleavage of thioredoxin under normal conditions was not feasible due to the extensive aggregation problems experienced when pheromaxein subunits exist separately. PheroA/trx and pheroC/trx were therefore mixed together and cleaved from thioredoxin simultaneously so that pheromaxein subunits were given an instant opportunity to associate under oxido-shuffling conditions. The glutathione oxido-shuffling system allowed the disulphide bridges between pheromaxein A and C to rearrange until the correct native structure was formed. This novel approach combines affinity purification with a coupled fusion protein-cleavage and refolding technique.     

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli

Abstract Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (lon protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.     

Construction of a high-copy "ATG vector" for expression in Escherichia coli.

We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.     

Independent and coupled translational initiation of atp genes in Escherichia coli: experiments using chromosomal and plasmid-borne lacZ fusions

The translational initiation rates directed by the translational initiation regions (TIRs) of the atpB, atpH, atpA and atpG genes of Escherichia coli were investigated using lacZ fusions present on plasmids as well as integrated into the chromosome. This was the first investigation of the translational efficiency of the atpB gene, whose unfused product (subunit a) can be toxic to the cell. The specific mRNA levels, rates of in vivo protein synthesis and beta-galactosidase activities encoded by the atp::lacZ fusions were compared in order to obtain valid estimates of relative translation rates. The results indicate that in the E. coli atp operon, translation directed by the atpB, atpH and atpG TIRs is less efficient than that directed by the atpA TIR, and are thus consistent with earlier measurements of direct atp gene expression. Initiation is, however, to differing extents, controlled by coupling to the translation of upstream neighbours. There is particularly tight coupling between atpH and atpA. Increasing the distance between these two genes whilst maintaining the original atpA TIR structure decreased the degree of coupling. The influence of manipulations of the atpG TIR structure upon translational efficiency was quantitatively more pronounced when the atpG fusions were present as a single copy per chromosome. This is likely to be related to the mRNA binding characteristics of 30S ribosomal subunits and/or to the influence of other (trans-acting) factors. The control of independent and coupled initiation at the atp TIRs is discussed in relation to mRNA structure and possible cis and trans regulatory phenomena.     

Improvement of nutritional value and functional properties of soybean glycinin by protein engineering

Glycinin is one of the predominant storage proteins of soybean. To improve its functional properties (heat-induced gelation and emulsification) and/or nutritional value, the A1aB1b proglycinin subunit was modified on the basis of genetically variable domains suggested from the comparison of amino acid sequences of glycinin-type globulins from various legumes and nonlegumes and the relationships between the structure and the functional properties of glycinin. Thus, nucleotide sequences corresponding to each of the variable domains were deleted from the cDNA encoding the A1aB1b proglycinin, and a synthetic DNA encoding four continuous methionines was inserted into the cDNA region corresponding to each of the variable domains. Expression plasmids carrying the modified cDNAs were constructed and expressed in Escherichia coli strain JM105. Some of the modified proteins were accumulated as soluble proteins in the cells at a high level and self-assembled. They exhibited functional properties superior to those of the native glycinin from soybean, which establishes the possibility of creating theoretically designed novel glycinins with high food qualities.     

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.     

Identification of marker proteins for the adulteration of meat products with soybean proteins by multidimensional liquid chromatography-tandem mass spectrometry

Soybean proteins are frequently added to processed meat products for economic reasons and to improve their functional properties. Monitoring of the addition of soybean protein to meat products is of high interest due to the existence of regulations forbidding or limiting the amount of soybean proteins that can be added during the processing of meat products. We have used chromatographic prefractionation on the protein level by perfusion liquid chromatography to isolate peaks of interest from extracts of soybean protein isolate (SPI) and of meat products containing SPI. After enzymatic digestion using trypsin, the collected fractions were analyzed by nanoflow liquid chromatography-tandem mass spectrometry. Several variants and subunits of the major seed proteins, glycinin and beta-conglycinin, were identified in SPI, along with two other proteins. In soybean-protein-containing meat samples, different glycinin A subunits could be identified from the peak discriminating between samples with and without soybean proteins added. Among those, glycinin G4 subunit A4 was consistently found in all samples. Consequently, this protein (subunit) can be used as a target for new analytical techniques in the course of identifying the addition of soybean protein to meat products.     

The control of lambda DNA terminase synthesis.

Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques.     

A system for dual protein expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.     

重组人血小板生成素在大肠杆菌中表达的研究

采用化学法全合成了编码人血小板生成素（ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ,ＴＰＯ）成熟肽Ｎ端１５３氨基酸的基因序列,构建基于该合成基因的表达质粒,结果以谷胱甘肽转硫酶－ＴＰＯ１５３（ＧＳＴ－ＴＰＯ１５３）融合蛋白的方式获得了占全菌蛋白４０％的高效表达．进一步采用ＰＣＲ方法分别对ＴＰＯ合成基因及ＴＰＯｃＤＮＡ的翻译起始区（ＴＩＲ）序列进行定点突变,以降低这一区域的Ｇ－Ｃ含量．将突变序列分别插入到ｐＢＶ２２０表达载体中,重组质粒在转化大肠杆菌ＪＭ１０９后,均获得了表达,其中ＴＩＲ区突变后的合成基因表达产物约占全菌蛋白的１５％．为研究基因下游结构对表达的影响,在不改变氨基酸组成的基础上,构建了ＴＰＯ合成基因与ＴＰＯｃＤＮＡ的杂合序列表达质粒．研究结果表明翻译起始效率是影响ｒｈ－ＴＰＯ在大肠杆菌中表达的重要因素之一,同时基因下游序列的组成对表达水平也会产生影响．     

Autogenous regulation of RNA polymerase beta subunit synthesis in vitro.

The effects of Escherichia coli RNA polymerase and its subassemblies and subunits on the in vitro synthesis of beta subunit directed by DNA from a lambda transducing phage lambdadrif+-6 were investigated. This phage carries the structural gene (rpoB) for beta subunit as well as the genes for EF (translation elongation factor)-Tu, some ribosomal proteins, and stable RNAs of the E. coli chromosome. Among the RNA polymerase proteins examined, the two oligomers, holoenzyme and alpha2beta complex, repressed the synthesis of only the beta subunit but not of other proteins encoded by the phage DNA. The results indicate that the expression of at least the betabeta' (rpoBC) operon is under autogenous regulation, in which both holoenzyme and alpha2beta complex function as regulatory molecules with repressor activity.