Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Heterologous protein expression using a novel stress-responsive protein of E. coli RpoA as fusion expression partner

E. coli proteome response to the stressor 2-HEDS was analyzed through two-dimensional gel electrophoresis (2-DE), and we identified DNA-directed RNA polymerase -subunit (RpoA) as stress-responsive protein. Even under stress situation where the total number of soluble proteins decreased by 9.8%, the synthesis level of RpoA was increased 1.5-fold. As a fusion expression partner as well as solubility enhancer, RpoA facilitated the folding and increased significantly the solubility of many aggregation-prone heterologous proteins (human minipro-insulin, human epidermal growth factor, human prepro-ghrelin, human interleukin-2, human activation induced cytidine deaminase, human glutamate decarboxylase, Pseudomonas putida cutinase, human ferritin light chain, human granulocyte colony-stimulating factor, and cold inflammatory syndrome1 protein Nacht domain) in E. coli cytoplasm. Due probably to intrinsic high folding efficiency and/or chaperone-like activity, RpoA was very effective in shielding interactive surfaces of heterologous proteins that are associated with non-specific protein–protein interaction leading to the formation of inclusion bodies. RpoA was also well suited for the production of biologically active fusion mutant of Pseudomonas putida cutinase that is of much biotechnological and commercial interest.     

Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repair-deficient Escherichia coli strain

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating agent widely used as an anticancer drug and also as an immunosuppressant. Its chemical structure and clinical experience indicate that CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to induce mutations and gene expression changes in the wild-type (WT) Escherichia coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E. coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA array method. The results showed that CLB is a potent mutagen in MV1932 cells compared with the E. coli WT strain AB1157, emphasizing the role of 3-methyladenine DNA glycosylases I and II in protecting the cells from CLB-induced DNA damage and subsequent mutations. Global gene expression profiling revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of 4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these MV1932 genes were downregulated, while 22% were upregulated in WT cells. The downregulated genes in MV1932 represented most (19/23) functional categories, and unexpectedly, many of them code for proteins responsible for genomic integrity. These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC (genetic recombination). Our results strongly suggest that in addition to the DNA damage itself, the downregulation of central protecting genes is responsible for the decreased cell survival (demonstrated in a previous work) and the increased mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.     

Deletion or alteration of hydrophobic amino acids at the first and the third transmembrane domains of hepatitis B surface antigen enhances its production in Escherichia coli

To investigate the failure of high-level production of hepatitis B viral (HBV) surface antigen (HBsAg), including three authentic forms, large (L), middle (M) and major/small (S) HBsAg, in Escherichia coli, we employed the high-expression vector pGEX containing the glutathione S-transferase-encoding gene (GST) to study HBsAg production. Different fragments of HBV DNA containing the entire pre-S1/pre-S2/S region (for L protein), or partial pre-S1, pre-S2, pre-S1/pre-S2 and pre-S2/S region (for M protein), were fused downstream from the GST gene, in order to obtain five plasmids which encode GST-HBsAg fusion proteins. SDS-PAGE analyses revealed that cells containing plasmids with a full-length S region (pGLS and pGMS) produced undetectable GST-HBsAg fusion proteins, in contrast to those cells harboring plasmids without the S region (pGS1, pGS2 and pGS1S2), which synthesized fusion proteins in 3–10% of the total cellular protein. Using an immunoblot method to screen HBsAg production in cells which harbored plasmids derived from exonuclease BAL 31-digested pGLS, we obtained eight positive clones. Nucleotide sequence analyses of plasmids from the positive clones revealed that termination, deletion or frameshift occurred at the regions encoding either the first or the third transmembrane domain of the major HBsAg. Correlation between the production level of GST-HBsAg fusion proteins and their constituent and arrangement of amino acids (aa) at the last 20 as among 15 clones suggested that the fusion protein ended with a longer stretch of or a higher ratio of hydrophobic as had a lower production in E. coli.     

Bacterial orthologues indicate the malarial plastid gene ycf24 is essential

ycf24 is a well conserved gene found in all major groups of bacteria, as well as on red algal plastid genomes and the vestigal plastid genome of apicomplexan pathogens like the malaria parasite Plasmodium falciparum (ORF470). Some database annotations describe Ycf24 as an ABC transporter subunit, but we find the level of significance is low. To investigate ycf24′s function we disrupted it in the cyanobacterium Synechocystis sp., strain PCC6803 which has a multi-copy genome. This showed ycf24 is essential, partial loss producing a terminal phenotype of chlorosis, reduced cell size, loss of DNA, and a striking arrest in cytokinesis. Attempts to disrupt the single copy of ycf24 in E. coli failed to give stable transformants. When Ycf24 was over-expressed in E. coli as a soluble fusion protein, it localized mostly as a band on either side of the nucleoid and nucleoid partitioning was aberrant. We propose the relict plastid organelle of apicomplexans retains its capacity for protein synthesis because Ycf24 is essential.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5′-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3′ end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

Extracellular secretion of STa heat-stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1–19) and Pro (aa 20–53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.     

Synthesis of the measles virus nucleoprotein in yeast Pichia pastoris and Saccharomyces cerevisiae

The development of a simple, efficient and cost-effective system for generation of measles virus nucleoprotein might help to upgrade reagents for measles serology. The gene encoding measles nucleoprotein was successfully expressed in two different yeast genera, Pichia pastoris and Saccharomyces cerevisiae, respectively. Both yeast genera synthesized a high level of nucleoprotein, up to 29 and 18% of total cell protein, in P. pastoris and S. cerevisiae, respectively. This protein is one of most abundantly expressed in yeast. After purification nucleocapsid-like particles (NLPs) derived from both yeast genera appeared to be similar to those detected in mammalian cells infected with measles virus. A spontaneous assembly of nucleoprotein into nucleocapsid-like particles in the absence of the viral leader RNA or viral proteins has been shown. Compartmentalisation of recombinant protein into large compact inclusions in the cytoplasm of yeast S. cerevisiae by green fluorescence protein (GFP) fusion has been demonstrated. Sera from measles patients reacted with the recombinant protein expressed in both yeast genera and a simple diagnostic assay to detect measles IgM could be designed on this basis.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备

为原核表达严重急性呼吸综合征冠状病毒2（简称新型冠状病毒,severe acute respiratory syndrome-coronavirus 2,SARS-CoV-2）S蛋白受体结合域（receptor binding domain, RBD）并制备多克隆抗体,利用基因克隆技术将RBD基因连接到原核表达载体pGEX-6p-1和pET-32a(+)上,电转化至大肠杆菌XL1-Blue感受态细胞,利用优化后的表达条件大量表达重组蛋白,经亲和层析纯化后通过SDS-PAGE检测蛋白的表达情况。利用GST-RBD融合蛋白作为免疫抗原免疫小鼠制备多克隆抗体,ELISA和Western blot分析抗血清的效价和特异性。PCR鉴定和序列测定结果显示,成功构建了重组载体pGEX-RBD和pET-RBD,在大肠杆菌中实现了GST-RBD和RBD-His融合蛋白的可溶性高效表达。研究获得的多克隆抗体的滴度达到约1∶3 000,并具有良好的结合特异性。原核表达的可溶性新型冠状病毒RBD重组蛋白具有良好的免疫原性,为后续制备基因工程抗体奠定了实验基础。     

Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HIyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA     

Rapid and efficient isolation of highly specific antibodies from an antiserum against a pool of proteins

Antibodies with desired specificity to proteins of interest provide important and versatile tools for detecting and localizing specific proteins in organisms. With the rapidly increasing number of genes cloned, the demand for antibodies to the gene products is increasing greatly. We developed a procedure to isolate highly specific antibodies to an insect intestinal mucin (IIM) from a polyclonal antiserum, which served as a “library of antibodies,” by using an E. coli lysate of the IIM cDNA clone. This procedure allows rapid and efficient isolation of target protein specific antibodies from a polyclonal antiserum made against a pool of antigens.     

Surface display of a glucose binding protein

Glucose binding protein (GBP) from Escherichia coli has been widely used to develop minimally invasive glucose biosensors for diabetics. To develop a cell-based glucose biosensor, it is essential to functionally display GBP on the cell surface. In this study, we designed a molecular structure to display GBP on the outer membrane of E. coli. We fused GBP with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli). With this molecular design, we have successfully displayed GBP on the surface of E. coli. Using FITC-conjugated Dextran, we demonstrated that glucose’s binding sites of surface-displayed GBP were accessible to glucose. Furthermore, we showed that glucose transport in a GBP-deficient E. coli NM303 could be restored by displaying GBP on the surface of NM303. 0.51 h⁻¹ of specific growth rate was attained for NM303/pESDG grown in M9 minimal medium supplemented with 2 g/l glucose, whereas no growth was observed for NM303 in the same medium. Both NM303 and NM303/pESDG grew in M9 medium supplemented with 1 mM of fucose. Because cell surface is an interface between intracellular and extracellular molecular events, this technique paves a way to develop cell-based glucose biosensors.