The ;heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the gene, nucleotide and amino acid sequence

The gene coding for the `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis was isolated in an expression vector. Expression of the heavy subunit in Escherichia coli was detected with antibodies raised against crystalline reaction centres. The entire subunit, and not a fusion protein, was expressed in E. coli. The protein coding region of the gene was sequenced and the amino acid sequence derived. Part of the amino acid sequence was confirmed by chemical sequence analysis of the protein. The heavy subunit consists of 258 amino acids and its mol. wt. is 28 345. It possesses one membrane-spanning α-helical segment, as was revealed by the concomitant X-ray structure analysis.     

Expression of constitutive and tissue-specific acyl carrier protein isoforms in Arabidopsis

We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hloušek-Radojčić, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.     

Gene Cloning, DNA Sequencing, and Expression of Thermostable β-Mannanase from Bacillus stearothermophilus

A DNA genomic library constructed from Bacillus stearothermophilus, a gram-positive, facultative thermophilic aerobe that secretes a thermostable β-mannanase, was screened for mannan hydrolytic activity. Recombinant β-mannanase activity was detected on the basis of the clearing of halos around Escherichia coli colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum. The nucleotide sequence of the mannanase gene, manF, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da. From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases. The manF coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in E. coli as a recombinant fusion protein containing a hexahistidine N-terminal sequence. The fusion protein has thermostability similar to the native enzyme and was purified by Ni²⁺ affinity chromatography. The values for the kinetic parameters V_max and K_m were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme.     

Characterization of a mitochondrially targeted single-stranded DNA-binding protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.     

Surface display of the HPV L1 capsid protein by the autotransporter Shigella IcsA

Autotransporters have become attractive tools for surface expression of foreign proteins in Gram-negative bacteria. In this study, the Shigella autotransporter IcsA, has been exploited to express the human papillomavirus (HPV) type 16 L1 capsid protein in Shigella sonnei and Escherichia coli. The L1 gene was fused in-frame to replace the coding sequence of the IcsA passenger domain that is responsible for actin-based motility. The resultant hybrid protein could be detected by an anti-L1 antibody on the surface of S. sonnei and E. coli. In E. coli, the protein was expressed on the entire surface of the bacterium. In contrast, the protein was detected mainly at one pole of the Shigella bacterium. However, the protein became evenly distributed on the surface of the Shigella bacterium when the icsP gene was removed. Our study demonstrated the possibility of exploiting autotransporters for surface expression of large, heterologous viral proteins, which may be a useful strategy for vaccine development.     

Bioaccumulation of heavy metals in Escherichia coli expressing an inducible synthetic human metallothionein gene

A synthetic DNA coding for human hepatic metallothionein (HMT), fraction MT-2, has been cloned in the plasmid vector pING 1 and expressed in E. coli. Upon induction with arabinose, an araB′-HMT fusion protein is synthesized. The fusion protein is produced to a level of approximately 8% of the total protein in E. coli, with an apparent half-life of 50 min. Without arabinose, no fusion protein is expressed, demonstrating that the expression system is tightly regulated. In the presence of ¹⁰⁹Cd, the fusion protein binds the metal ion in vitro. Concurrently, a direct correlation is found between the expression in E. coli of the araB′-HMT fusion protein which binds Cd²⁺ in vitro and the bioaccumulation of Cd²⁺ in vivo. The bioaccumulation of Cu⁺ in E. coli, when the fusion protein is produced, has also been established.     

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe³⁺ had an active effect on the enzyme obviously.     

Fusion expression of mutated cecropin CMIV inE. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

Simple and Efficient Method for Heterologous Expression of Clostridial Proteins

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.     

Expression of a recombinant Human papillomavirus 16 E6GT oncoprotein fused to N- and C-termini of Potato virus X coat protein in Nicotiana benthamiana

Infection with Human papillomaviruses (HPVs) is linked to cervical cancer, which is one of the most common cancers found among women worldwide. Despite preventive immunization, a therapeutic vaccine, targeting infected individuals, is required. Vaccine strategies for treatment of HPV—induced cervical cancer are based on E7 and E6 oncoproteins. In this work we report the transient expression of chimeric particles containing the E6 oncoprotein from Human papillomavirus type 16 (HPV-16) in plants. We fused a mutagenized coding sequence of the E6 oncoprotein (E6GT) with the coding sequence of Potato virus X coat protein (PVX CP) both with the 5′- and 3′-terminus using linkers of different length (0, 4 or 15 amino acids). The expression in E. coli was performed to assess the characteristics of the recombinant protein prior to plant expression. The yield and immunological reactivity of the expressed proteins were screened with anti-PVX CP and E6 antibodies. The highest yields of chimeric particles were observed in the transgenic N. benthamiana expressing Potato virus A HC-Pro protein and the Tobacco mosaic virus movement protein. When inoculated on host plants, these recombinant viruses were not able to spread systemically. The obtained results revealed the new relations for design of expression cassettes for plant-based vaccine production.     

金黄色葡萄球菌肠毒素 C2 的基因克隆、 表达及其生物学活性

利用 PCR 技术从金黄色葡萄球菌的基因组 DNA 中克隆 SEC2 全长基因, PCR 产物与 pGEM-T 载体连接,经测序证实后进行亚克隆,构建其表达载体 pET-28a-SEC2 ,在大肠杆菌 BL21(DE3) 中表达成熟重组蛋白 (rSEC2) , 纯化 rSEC2 蛋白并对其生物学活性进行研究 . 结果表明：成功克隆了 SEC2 全长基因,测序证实该基因共 717 bp ,编码 239 个氨基酸,与 GenBank 中收录的 SEC2 成熟蛋白质序列完全一致, SEC2 基因登录 GenBank(Accession number ： AY450554) ; 构建了 SEC2 的表达载体 pET-28a-SEC2 ,并在大肠杆菌 BL21(DE3) 中得到高效可溶性表达,可溶性的 rSEC2 经 Ni²⁺ 亲和层析纯化达到电泳纯,纯化的 rSEC2 蛋白经蛋白质印迹检测,并能有效刺激人外周血单个核细胞的增殖,被 rSEC2 刺激的外周血单个核细胞在体外对肿瘤细胞的生长有显著的抑制作用 .     

The translation of recombinant proteins in E. coli can be improved by in silico generating and screening random libraries of a −70/+96 mRNA region with respect to the translation initiation codon

Recombinant protein translation in Escherichia coli may be limited by stable (i.e. low free energy) secondary structures in the mRNA translation initiation region. To circumvent this issue, we have set-up a computer tool called ‘ExEnSo’ (Expression Enhancer Software) that generates a random library of 8192 sequences, calculates the free energy of secondary structures of each sequence in the −70/+96 region (base 1 is the translation initiation codon), and then selects the sequence having the highest free energy. The software uses this ‘optimized’ sequence to create a 5′ primer that can be used in PCR experiments to amplify the coding sequence of interest prior to sub-cloning into a prokaryotic expression vector. In this article, we report how ExEnSo was set-up and the results obtained with nine coding sequences with low expression levels in E. coli. The free energy of the −70/+96 region of all these coding sequences was increased compared to the non-optimized sequences. Moreover, the protein expression of eight out of nine of these coding sequences was increased in E. coli, indicating a good correlation between in silico and in vivo results. ExEnSo is available as a free online tool.     

Cloning,sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme

The bovine pancreatic (bp-) DNase I gene has been cloned from bp-cDNA and expressed in E. coli. A polynucleotide sequence of 1295 base pairs was deduced from clones of the cDNA. The sequence showed an open reading frame which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pET-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleotide coding for Met–Ala at the 5′-end. The plasmid was transformed into E. coli strain DH5α and the active recombinant bovine (rb-) DNase I was produced after induction of protein synthesis. From the induced culture medium, rb-DNase I was purified by chromatography on a Mono Q column. The purified rb-DNase I showed a molecular mass of 29 kDa and had the same specific activity as bp-DNase I. The NH₂-terminus of rb-DNase I was Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bp-DNase I, Asn was not glycosylated.     

Overproduction of Biologically-active Human Nerve Growth Factor in Escherichia coli

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the β-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the β-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.     

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities

The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC₅₀?=?13–15 μg/ml).     

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

High-level soluble expression of hIGF-1 fusion protein in recombinant Escherichia coli

Human insulin-like growth factor 1 (hIGF-1) is one kind of growth factor with clinical significance in medicine. The expression of TrxA-hIGF-1 fusion protein was rationally compared in three different Escherichia coli hosts (BL21 (DE3), Rosetta (DE3) and Rosetta-gami (DE3)) with the transformation of plasmid pET32-hIGF-1. The highest productivity of soluble hIGF-1 fusion protein was achieved in E. coli Rosetta-gami (DE3). Moreover, the effects of different expression conditions in this E. coli Rosetta-gami (DE3)/pET32-hIGF-1 host were systematically investigated to improve the expression level of the fusion protein. Under the optimized conditions, a high percent of the target fusion protein (96%) was expressed as soluble form with the volumetric production of soluble fusion protein reaching up to 2.06 g/L. After cell disruption, the soluble fusion protein was separated effectively by affinity chromatography and cleaved by enterokinase, with the concentration of mature hIGF-1 reaching up to 0.42 g/L in the mixture. The present work should be useful for the enhanced production of soluble protein with multiple disulfide bonds in E. coli.