• Journal of Microbiology and Biotechnology
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• 제7권4호
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.244-252
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• 2007

Escherichia coli has a PhoR-PhoB two-component regulatory system to detect and respond to the changes of environmental phosphate concentration. For the E. coli W3110 strain growing under phosphate-limiting condition, the changes of global gene expression levels were investigated by using DNA microarray analysis. The expression levels of some genes that are involved in phosphate metabolism were increased as phosphate became limited, whereas those of the genes involved in ribosomal protein or amino acid metabolism were decreased, owing to the stationary phase response. The upregulated genes could be divided into temporarily and permanently inducible genes by phosphate starvation. At the peak point showing the highest expression levels of the phoB and phoR genes under phosphate-limiting condition, the phoB- and/or phoR-dependent regulatory mechanisms were investigated in detail by comparing the gene expression levels among the wild-type and phoB and/or phoR mutant strains. Overall, the phoB mutation was epistatic over the phoR mutation. It was found that PhoBR and PhoB were responsible for the upregulation of the phosphonate or glycerol phosphate metabolism and high-affinity phosphate transport system, respectively. These results show the complex regulation by the PhoR-PhoB two-component regulatory system in E. coli.

• 한국미생물·생명공학회지
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• 제20권6호
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• pp.637-641
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• 1992

한국 재래식 된장.간장 발효균 Bacillus sp. SSA3 균주의 expression vector를 개발하기 위해 Bacillus sp. SSA3의 chromosomal DNA로부터 유전자의 promoter 부위를 cloning 하였다. Recombinant plasmid를 제작하기 위하여 Bacillus sp. SSA3의 chromosomal DNA을 HindIII로 절단한 단편을 pGR71 plasmid의 CAT gene과 pUC18 plasmid의 $\beta$-galactosidase gene의 전방에 삽입시킨 후, E.coli JM109에 형질전환하였다.E. coli JM109의 chloramphenicol 내성 clones으로부터 6 recombinant plasmid를 선별하였다. 이들 선별된 plasmid는 Bacillus sp. SSA3의 expression vector로 사용시 각 재조합 plasmid 중에 삽입된 promoter의 염에 대한 영향의 정도를확인하기 위해 10% NaCl이 첨가된 LB medium상에서 배양하였을 때, 이들 중 Bacillus sp. SSA3의 4 clones은 융합 CAT gene의 발현이 강하게 감소되었으나 2 clones은 약하게 저해되었다.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.196-205
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• 2015

A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.225-228
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• 2001

RhRnBp and Peysn5 are the proteins related to carbohydrate synthesis. RhRnBp originated form human was expressed as inclusion body in E. coli. Peysn5 originated form actinomadura was expressed as low level and inclusion body in E. coli. Ub, Trx, MalE and NusA is used as fusion partner to RhRnBp and Peysn5. The solubility of all fusion protein is NusA > MalE> Trx > Ub. Expression level of RhRnBp fusions in $37^{\circ}C$ is higher than that in $25^{\circ}C$. However in the case of Peysn5. Expression levels in $25^{\circ}C$ were higher. MalE fusion had highest activity in RhRnBp fusions. There were no activity in Peysn5.

• Journal of Microbiology
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• 제34권1호
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• pp.15-22
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• 1996

The cloned X-bacterial gene (groEx) which is analogous to groE of E. Coli strongly expressed in E. coli when grown at the temperature 27.deg. C or higher without having to add any inducers. By S1-nuclease mapping, primer extension analysis and site directed mutagenesis, we found 4 promoters in the gene. Among them two promoters located at 5'-extended region of the gene are homologous to the promoters found in groE family of heat-shock genes ; they are , .sigma.$^{32}$ factor-dependent P1 promotor and .delta$^{70}$factor-dependet P2 promoter. The other two promoters found within the coding region of groESx were P3, 5'-TTGGCG-(18 bases)-AATACT-3' and P4, 5'-TTGGCA-(19 bases)-TAAGT which overlapped within 49 bases. These unique intragenic .delta.$^{70}$-dependent promoters are the first to be cloned and characterized in groE analogous heat-shock genes so far. These P3 and P4 promoters appeared to be responsible for the strong expression of GroElx in X-bacteria in vivo.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.620-623
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• 2003

The delta sleep-inducing peptides (DSIP, Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu) is an important regulatory hormone, controlling hypothalamus and pituitary functions. In the current study, an expression system was designed for the rapid and economic expression oi recombinant DSIP for biophysical studies. Artificially synthesized oligonucleotides encoding DSIP were cloned into a pGEX-KG vector and expressed in E. coli (BL21). The recombinant GST-DSIP was then readily purified using a GST affinity column. To obtain intact DSIP from the GST-DSIP, thrombin cleavage and a CNBr reaction were successively carried out. The DSIP in the CNBr reaction mixture was subjected to RP-HPLC purification to yield 1.2 mg DSIP from a 1 liter culture of E. coli. Identification of the DSIP was peformed using MALDI-MS and an amino acid composition analysis.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.753-759
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• 2002

$\beta$-Cyclodextrin glucanotransferase ($\beta$-CGTase) was overproduced extracellularly using recombinant E. coli by transforming the plasmid pECGT harboring a secretive signal peptide. The $\beta$-CGTase gene of alkalophilic Bacillus firmus var alkalophilus was inserted into the high expression vector pET20b(+) containing a secretive pelB signal peptide, and then transformed into E. coli BL2l(DE3)pLysS. The optimum culture conditions fer the overproduction of $\beta$-CGTase were determined to be TB medium containing 0.5% (w/v) soluble starch at post-induction temperature of $25^{\circ}C$. A significant amount of $\beta$-CGTase, up to 5.83 U/ml, which was nine times higher than that in the parent strain B. firmus var. alkalophilus, was overproduced in the extracellular compartment. A pH-stat fed-batch cultivation of the recombinant E. coli was also performed to achieve the secretive overproduction of $\beta$-CGTase at a high cell density, resulting in production of up to 21.6 U/ml of $\beta$-CGTase.

• Journal of Microbiology and Biotechnology
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• 제27권5호
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• pp.965-974
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• 2017

The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and response surface methodology, under which the yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature, and particle diameter size were 1.01E-07 M, $55.2{\pm}0.3^{\circ}C$, and 9.388 nm for the scFv expressed by B. choshinensis, and 4.53E-07 M, $52.5{\pm}0.3^{\circ}C$, and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1448-1452
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• 2006

The effects of amplifying the gluconeogenic phosphoenolpyruvate carboxykinase of Escherichia coli ($pck_{Ec}$) on succinic acid production in E. coli were examined under anaerobic condition. No significant increase in succinic acid production was observed in E. coli overexpressing the $pck_{Ec}$ gene without supplementing $NaHCO_{3}$ or $MgCO_{3}$. On the other hand, succinic acid production was enhanced as the $NaHCO_{3}$ concentration was increased. When 20 g/l of $NaHCO_{3}$ was added, succinic acid production in recombinant E. coli overexpressing PCK was 2.2-fold higher than that observed in the wild-type strain. It was concluded that the gluconeogenic $pck_{Ec}$ overexpression enabled E. coli to enhance succinic acid production only under the high bicarbonate supplementation condition.