Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

Cloning and Expression of HPV18 E6 Gene / 天津医药

Objective: To express the protein of HPV18E6 based on pET-32a(+) at high level and study the expression and significance of HPV18E6 proteins in laryngeal carcinoma. Methods: The HPV18E6 gene was amplified by PCR and cloned into pET-32a(+). The amplified fragment was inserted into the plasmid pET32a (+) that was digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET32/E6 was transformed into E.coli JM109 which was selected with ampicillin. The recombinant plasmids were successfully introduced into E.coli BL21(DE 3) and were induced by IPTG. SDS-PAGE and Western blot analysis were used to detect the confusion protein. Finally, the optimization of expression conditions, such as temperature, concentration of IPTG, was studied. Results: The recombinant plasmids were identified and confirmed with enzyme digestion and sequencing. The BL21(DE3) transformed recombinant plasmid pET32/E6 had expressed HPV18E6 recombinant protein effectively. The optimum conditions of expression were 37 ℃, 1 mmol/L IPTG. Conclusion:Prokaryotic expression vector pET-32a(+)-HPV18E6 was successfully constructed. The high-level expression of HPV18E6 was achieved in E.coli BL21(DE3).

Prokaryotic 16S rDNAs from Various Prokaryotes That Are Suggested as Etiologies of Idiopathic Chronic Prostatitis / 대한비뇨기과학회지

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.

Cloning and prokaryotic expression of a novel binding protein 1 of HBeAg / 解放军医学杂志

Objective To clone the human gene of Hepatitis B virus e antigen binding protein 1 (HBEBP1), which was screened with yeast two-hybrid system and bioinformatics techniques, to construct prokaryotic expression vector of pET-32a(+)-HBEBP1, and to induce the expression of recombinant protein in E. coli BL21. Methods The DNA fragment of HBEBP1 of about 372bp was amplified by reverse transcription PCR (RT-PCR), in which the mRNA was taken from HepG2 cells as the template, and cloned into pGEM-T vector. After restriction enzyme digestion identification and sequencing, the correct target DNA fragment was inserted into inducible prokaryotic expression vector pET-32a(+) and then transformed into competent E. coli BL21. By restriction enzyme digestion and PCR, the positive transformed clones were identified and induced with IPTG to obtain fusion protein. The HBEBP1 fusion protein was analyzed by Western blotting hybridization. Results The 372bp DNA fragment of HBEBP1 was amplified by RT-PCR. The recombinant expression vector pET-32a(+)-HBEBP1 was constructed successfully. After transformation with pET-32a(+)-HBEBP1 and induction with IPTG, the recombinant target protein of about 33kD was obtained, which was consistent with our anticipation. Western blotting assay showed that the protein had good specificity. Conclusions The recombinant prokaryotic expression vector pET-32a(+)-HBEBP1 is constructed, and the HBEBP1 gene is cloned successfully. The HBEBP1 fusion protein could be expressed in prokaryotic expression system of E. coli. These results lays a foundative for studying the immunogenicity and the biological characteristics of the HBEBP1 protein.