• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.228-233
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• 2008

Effects of chaperones on mRNA stability and gene expression were studied in order to develop an efficient Escherichia coli expression system that can maximize gene expression. The stability of mRNA was modulated by introducing various secondary structures at the 5'-end of mRNA. Four vector systems providing different 5'-end structures were constructed, and genes encoding GFPuv and endoxylanase were cloned into the four vector systems. Primer extension assay revealed different mRNA half-lives depending on the 5'-end secondary structures of mRNA. In addition to the stem-loop structure at the 5'-end of mRNA, coexpression of dnaK-dnaJ-grpE or groEL-groES, representative heat-shock genes in E. coli, increased the mRNA stability and the level of gene expression further, even though the degree of stabilization was varied. Our work suggests that some of the heat-shock proteins can function as mRNA stabilizers as well s protein chaperones.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.309.2-309.2
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• 2002

The FimH subunit of type l-fimbriated Escherichia coli has been determined as a major cause of urinary tract infection. To produce a possible vaccine antigen against urinary tract infection, the fimH gene was genetically coupled to the ctxa2b gene, which was then cloned into pMAL -p2E expression vector. The chimaeric construction of pMALfimH/ctxa2b was transformed into Escherichia coli TB1 and its N-terminal amino acid sequence was analyzed. (omitted)

• Microbiology and Biotechnology Letters
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• v.18 no.3
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• pp.305-308
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• 1990

In order to clone the gene coding for 3-isopropylmalate dehydrogenase of Muyveromyces fragilis, a shuttle plasmid vector pHNll4 was used. It can serve as a cloning vector in Saccharomyces cerevisiae DBY746 for other Sau3AI-cleaved DNA segment of Kluyveromyces fragilis. Two cloned fragments which complement the leu2 mutation of Saccharomyces cerevisiae and E, coli were obtained. Their length was 4.4 kb an 3.5 kb, and their orientation was opposite each other. From the fact that the two recombinant plasmids were expressed in Saccharomyces cerevisiae and E, coli, probably the two inserts had the promoter of Ktuyveromyces fi-agilis and that of Kluyveromyces fiagilis was efficiently assosiated with RNA polymerase of Saccharomyces cerevisiae and E. coli. According to the result of Southern hybridization, we thought that the cloned fragment has low homology with 3-isopropylmalate dehydrogenase coding region of E. coli and Saccharomyces cerevisiae.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.171-175
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• 2003

The putative endochitinase gene, yheB of Escherichia coli K-12 is not expressed under lab culture conditions. The endochitinase gene was amplified by PCR and subcloned into pET28c vector and pQE9 vector, respectively. The endochitinase produced in E. coli harboring pET28c containing yheB or pQE9 vector containing yheE was partly released into the growth medium. The overproduced endochitinase was partially purified by His affinity column chromatography and DE-52 column chromatography. The apparent molecular weight of the endochitinase determined by SDS-polyacrylamide gel electrophoresis was about 97,000. The purified E. coli endochitinase showed maximal chitinolytic activity at pH 6 and $40^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.161-168
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• 1986

A 1.95Kb Sau3Al fragment coding for $\alpha$-amylase from Bacillus amyloliquefaciens was isolated by the shotgun method using Escherichia coli as a host. The genome of Bacillus amyloliquefaciens was partially digested with the restriction endonuclease Sau3Al and joined to plasmid YRp7 cleaved with the restriction endonuclease BamHI. The $\alpha$-amylase gene present in a 1.95Kb insert was stably maintained and expressed in Escherichia coli. The amount of $\alpha$-amylase activity produced by Escherichia coli containing the hybrid plasmid pEA24 was about 65% of the activity produced by the donor Bacillus amyloliquefaciens strain. The properties of $\alpha$-amylase produced by Escherichia coli were very similar to those produced by Bacillus amyloliquefaciens as based on optimum temperature, pH, and effect of CaCl$_2$ concentration. About 70% of the $\alpha$-amylase produced by Escherichia coli was localized in the periplasmic space, whereas the remaining enzyme was localized in the inner part of the cell.

• Biomedical Science Letters
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• v.18 no.1
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• pp.29-34
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• 2012

Streptomycetes are gram-positive filamentous bacteria that are well-known for producing a vast array of bioactive compounds, including more than 70 % of commercially important antibiotics. For the research about Streptomyces sp., the protoplast and electroporation transformation method have been the general techniques for the construction of transformants. However, these techniques have low efficiency and are time-consuming. Another option is intergenic conjugation, which is used for DNA transfer using methylation-deficient E. coli as a DNA donor to avoid the methylated-DNA-dependent restriction systems of actinomycetes. This conjugation method has been widely improved and applied to many other actinomycetes. In this research, an effective transformation procedure for the construction of expression vector by using gateway system was established to avoid limit of restriction enzyme site for cloning of target gene based on transconjugation by Escherichia coli ET12567/pUZ8002 with a pSET152 integration vector.

• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.4
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• pp.301-305
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• 2001

The co-expression of the arg U gene in a double-vector expression system of recombi-nant Escherichia coli BL22(DE3)[pET-IEN2a+pAC-argU] significantly enhanced the production level of reconminant human interferon -$\alpha$2a(rhIFN-$\alpha$2a) in high cell density cultures, compared to a recombinant E. coli culture containing only the single expression vector, pET-IEN2a. The dry cell mass concentration increased to almost 100 g/L, and more than 4 g/L of rhIFN-$\alpha$2a was accumu-lated in the culture broth. Evidently, the synthesis of rhIFN-$\alpha$2a was strongly dependent on the pre-induction growtih rate and more efficient at a higher specific growth rate. The additional sup-ply of tRN $A^{Arg(AGG/AGA)}$ enhanced the expression level of the rhIFN-$\alpha$2a gene in the early stage of the post-induction phase, yet thereafter the specific production rate of rhIFN-$\alpha$2a rapidly de-creased due to severe segregational instability of plasmid vector pET-IEN2a. It would appear that the plasmid instability with only occurred to pET-IEN2a in the double vector system, was re-lated to the effect of translational stress due to the over expression of rhIFN-$\alpha$2a.

• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.491-494
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• 2009

Within the secondary metabolite class of flavonoids, which consist of more than 10,000 known structures, flavones define one of the largest subgroups. The diverse function of flavones in plants as well as their various roles in the interaction with other organisms offers many potential applications including in human nutrition and pharmacology. We used two genes, flavone synthase (PFNS-l) that converts naringenin into apigenin and flavone 7-O-methyltransferase (POMT-7) that converts apigenin into 7-O-methyl apigenin, to synthesize 7-O-methyl apigenenin from naringenin. The PFNS-l gene was subcloned into the E. coli expression vector pGEX and POMT-7 was subcloned into the pRSF vector. Since both constructs contain different replication origins and selection markers, they were cotransformed into E. coli. Using E. coli transformants harboring both PFNS-l and POMT-7, naringenin could be converted into 7-O-methyl apigenin, genkwanin.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.1-11
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• 2001

Lactic acid bacteria (LAB) are widely used for various food fermentation. With the recent advances in modern biotechnology, a variety of bio-products with the high economic values have been produced using microorganisms. For molecular cloning and expression studies on the gene of interest, E. coli has been widely used mainly because vector systems are fully developed. Most plasmid vectors currently used for E, coli carry antibiotic-resistant markers. As it is generally believed that the antibiotic resistance markers are potentially transferred to other bacteria, application of the plasmid vectors carrying antibiotic resistance genes as selection markers should be avoided, especially for human consump-tion. By contrast, as LAB have some desirable traits such that the they are GRAS(generally recognized as safe), able to secrete gene products out of cell, and their low protease activities, they are regarded as an ideal organism for the genetic manipulation, including cloning and expression of homologous and heterologous genes. However, the vec-tor systems established for LAB are stil insufficient to over-produce gene products, stably, limiting the use of these organisms for industrial applications. For a past decade, the two popular plasmid vectors, pAM$\beta$1 of Streptococcus faecalis and pGK12 theB. subtilis-E. coli shuttle vector derived from pWV01 of Lactococcus lactis ssp. cremoris wg 2, were most widely used to construct efficient chimeric vectors to be stably maintained in many industrial strains of LAB. Currently, non-antibiotic markers such as nisin resistance($Nis^{r}$ ) are explored for selecting recombi-nant clone. In addition, a gene encoding S-layer protein, slp/A, on bacterial cell wall was successfully recombined with the proper LAB vectors LAB vectors for excretion of the heterologous gene product from LAB Many food-grade host vec-tor systems were successfully developed, which allowed stable integration of multiple plasmid copies in the vec-mosome of LAB. More recently, an integration vector system based on the site-specific integration apparatus of temperate lactococcal bacteriophage, containing the integrase gene(int) and phage attachment site(attP), was pub-lished. In conclusion, when various vector system, which are maintain stably and expressed strongly in LAB, are developed, lost of such food products as enzymes, pharmaceuticals, bioactive food ingredients for human consump-tion would be produced at a full scale in LAB.