With the rapid development of genome sequencing technologies, a large amount of prokaryote genomes have been sequenced in recent years. To further investigate the models and functions of genomes, the algorithms for genome annotations based on the sequence and homology features have been widely implemented to newly sequenced genomes. However, gene annotations only using the genomic information are prone to errors, such as the incorrect N-terminals and pseudogenes. It is even harder to provide reasonable annotating results in the case of the poor genome sequencing results. The transcriptomics based on the technologies such as microarray and RNA-seq and the proteomics based on the MS/MS have been used widely to identify the gene products with high throughput and high sensitivity, providing the powerful tools for the verification and correction of annotated genome. Compared with transcriptomics, proteomics can generate the protein list for the expressed genes in the samples or cells without any confusion of the non-coding RNA, leading the proteogenomics an important basis for the genome annotations in prokaryotes. In this paper, we first described the traditional genome annotation algorithms and pointed out the shortcomings. Then we summarized the advantages of proteomics in the genome annotations and reviewed the progress of proteogenomics in prokaryotes. Finally we discussed the challenges and strategies in the data analyses and potential solutions for the developments of proteogenomics.
Genomics ; Molecular Sequence Annotation ; Prokaryotic Cells ; metabolism ; Proteomics ; Tandem Mass Spectrometry

OBJECTIVE: To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. METHODS: The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. RESULTS: The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. CONCLUSION We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
Animals ; Antibodies ; Capsid ; Capsid Proteins/genetics* ; Dependovirus/genetics* ; Prokaryotic Cells ; Rabbits ; Recombinant Proteins/genetics*

Although superoxide anions (O2.-) and H2O2 are generally considered to be toxic by-products of respiration, recent evidence suggests that the production of these reactive oxygen species (ROS) might be an integral component of membrane receptor signaling. In mammalian cells, a variety of extracellular stimuli have recently been shown to induce a transient increase in the intracellular concentration of ROS, and specific inhibition of the ROS generation resulted in a complete blockage of stimulant-dependent signaling. In the next few years, therefore, a flurry of research activity is expected in relation to the elucidation of ROS production in response to receptor stimulation, identification of ROS target molecules, and investigation of ROS elimination. The goal of this report is to review our current knowledge of ROS-regulated signal transduction and propose future directions.
Animal ; Eukaryotic Cells/metabolism ; Human ; Hydrogen Peroxide/metabolism* ; Oxidation-Reduction ; Prokaryotic Cells/metabolism ; Reactive Oxygen Species/metabolism ; Signal Transduction*

OBJECTIVETo investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells.

METHODSEukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity.

RESULTSThe Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction.

CONCLUSIONThe IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.

Animals ; Eukaryotic Cells ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation ; Hepatitis B ; metabolism ; Interferon-alpha ; biosynthesis ; genetics ; physiology ; Marmota ; metabolism ; Prokaryotic Cells ; metabolism ; Signal Transduction ; Transcription Factors

OBJECTIVETo construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.

METHODSPrimers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.

RESULTThe cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.

CONCLUSIONThe ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Antibodies, Monoclonal ; Biotechnology ; methods ; Cells, Cultured ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Lipopolysaccharide Receptors ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics

PURPOSE: Specific microorganisms, such as C trachomatis, Mycoplasma and T vaginalis, are rarely detected in idiopathic chronic prostatitis. However, fastidious and nonculturable microorganisms may be important in the etiology of idiopathic chronic prostatitis. The object of this study was to test a new PCR primer set to detect 16S rDNA from various prokaryotes suggestive of the etiologies of idiopathic chronic prostatitis. MATERIALS AND METHODS: A new 16S rDNA primer set was designed from common prokaryotic genetic sequences using bioinformatic tools. The genomic DNAs from E-coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Actinobacter baumanmii, coagulase negative Staphylococcus, Corynebacterium, Serratia marcescens, Proteus mirabilis and Staphylococcus aureus were extracted by boiling colonies from their plated cultures. The template DNAs from the above microorganisms were amplified using this new 16S rDNA primer set. RESULTS: The correct PCR product, 470 bp, was obtained from E-coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Actinobactrer baumanmii, Corynebacterium spp, Serratia marcescens and Proteus mirabilis. However, a constant result from gram positive bacteria, such as Stapylococci, could not be obtained. CONCLUSIONS: A new PCR primer set, which can detect various prokaryotes suggestive of the etiologies of idiopathic chronic prostatitis, was obtained.
Coagulase ; Corynebacterium ; DNA ; DNA, Ribosomal* ; Enterobacter cloacae ; Gram-Positive Bacteria ; Klebsiella pneumoniae ; Mycoplasma ; Polymerase Chain Reaction ; Prokaryotic Cells ; Prostatitis* ; Proteus mirabilis ; Pseudomonas aeruginosa ; Serratia marcescens ; Staphylococcus ; Staphylococcus aureus

To purify the extracellular domain of HER2 in vitro and improve its prokaryotic expression abundance, the cDNA fragment encoding extracellular domain of HER2 was obtained by PCR and cloned into the expression vector pGEX-6P-1. After transforming it into Escherichia coli BL21, we instituted an investigation of different inducing conditions to try out the optimal condition for expressing soluble fusion protein. As for insoluble inclusion bodies, they were dissolved in 8 M Urea and refolded in refolding buffer. The soluble protein and the refolded protein were purified with Glutathione Sepharose 4B, respectively. The results showed that both the soluble and insoluble protein existed in Escherichia coli, but the majority was insoluble. It is beneficial to the expression of soluble fusion protein by induction at lower temperature (30 degrees C) and higher optical density (A600= 1.8) with the use of certain additive in medium. By purification of the supernatant of the lysate and refolded protein, the yield of the fusion protein was about 1.23 mg per liter culture. As a result, we have obtained the maximum soluble extracellular domain of HER2 protein, and thus have laid a foundation for further work on functional study and antibody preparation for HER2.
Cloning, Molecular ; Escherichia coli ; genetics ; Genes, erbB-2 ; genetics ; Humans ; Prokaryotic Cells ; metabolism ; Protein Folding ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E. coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a (+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E. coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD= 0.992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Neisseria gonorrhoeae ; genetics ; Plasmids ; biosynthesis ; genetics ; Porins ; biosynthesis ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection ; Vaccines, Synthetic

To express recombinant Ancylostoma caninum anticoagulant peptide-c2 (AcAPc2), a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc2; positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 (about 30.1% fusion protein in total protein). AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by beta-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS-PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.
Animals ; Dogs ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genes, Helminth ; Genetic Vectors ; Helminth Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; RNA Splicing ; Recombinant Fusion Proteins ; chemistry ; pharmacology

In order to clone lvgA gene (Legionella virulence gene) of Legionella pneumophila and detect its expression in prokaryotic cell, we amplified the lvgA gene from the total cell DNA of Legionella pneumophila with PCR,and then inserted it into the coloning vector pUC18. The recombinant plasmid pUlvgA was obtained. After the recombinant plasmid pUlvgA was identified with restriction enzyme analysis, polymerase chain reaction and nucleotide sequencing analysis, the lvgA gene was subcloned into the prokaryotic expression vector pGEX-4T-1. The prokaryotic expression recombinant plasmid pGlvgA was constructed. After IPTG induction, the E. coli JM109 containing the recombinant plasmid pGlvgA expressed fusion protein. The expression of lvgA was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The results indicated that the lvgA gene of 627 bp long was amplified, the recombinant plasmids pUlvgA and pGlvgA were constructed successfully, and the GST-LvgA fusion protein of approximately 36 KDa in size was expressed in prokaryotic cell efficiently as expected.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Cytoplasm ; metabolism ; Escherichia coli ; metabolism ; Legionella pneumophila ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Virulence Factors ; biosynthesis ; genetics