• 산업기술연구
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• 제28권B호
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.357-366
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• 2019

We first confirmed the involvement of MalQ (4-${\alpha}$-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ${\Delta}malQ$, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-${\beta}$-CD: G4-${\beta}$-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.

• 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 Pharmaceutical Investigation
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• 제8권1호
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• pp.1-5
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• 1978

Drug interaction of a new antibiotic, methampicillin lysinate (MAL) with nine drugs were investigated using four species of gram positive and gram negative bacteria. The experimental results were as follows: 1. MIC of MAL were found to be decreased against E. coil when combined with mefenamic acid, probenecid, aluminium hydroxide gel or corticosteroids. The other drugs did not affect MIC of MAL against the same bacteria. 2. MIC of MAL were found to be increased against Staphylococcus aureus ATCC 6538-P, 9441 when combined with mefenamic acid, aluminum hydroxide gel or dexamethasone acetate. The other drugs did not affect MIC of MAL against the same bacteria. 3. MIC of MAL were found to be increased against Shigella dysenteriae when either of the nine drugs was combined. 4. MIC of MAL were found to be increased approximately 2.5 times when combined with Streptokinase-Streptodornase or hydrocortisone and to be decreased approximately 2 times when combined with probenecid or dexamethasone against Salmonella typhi(type 2). It seems the other drugs do not affect the MIC of MAL against the same bacteria.

• Archives of Pharmacal Research
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• 제22권3호
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• pp.274-278
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• 1999

Expression of the active urease of the enterobacterium, Klebsiella aerogens, requires the presence of the accessory genes (ureD, ureE, ureF, and ureG) in addition to the three structural genes (ureA, ureB, and ureC). These accessory genes are involved in functional assembly of the nickel-metallocenter for the enzyme. Characterization of ureF gene has been hindered, however, since the UreF protein is produced in only minute amount compared to other urease gene products. In order to overexpress the ureF gene, a recombinant pMAL-UreF plasmid was constructed from which the UreF was produced as a fusion with maltose-binding protein. The MBP-UreF fusion protein was purified by using an amylose-affinity column chromatography followed by an anion exchange column chromatography. Polyclonal antibodies raised against the fusion protein were purified and shown to specifically recognize both MBP and UreF peptides. The UreF protein was shown to be unstable when separated from MBP by digestion with factor Xa.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1503-1509
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• 2014

With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 ($pyrE_{sso}$) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an ${\alpha}$-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an ${\alpha}$-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase ($gdhA_{saci}$) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The ${\alpha}$-glucosidase activity was confirmed by the hydrolysis of $pNP{\alpha}G$. The pINEX vector should be applicable in delineating gene functions in this organism.

• ALGAE
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• 제19권2호
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• pp.145-148
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• 2004

As a result of the 2-year monthly monitoring of the phytoplankton community at 3 stations in Mankyeong Estuary, Korea, we learned that cyan bacterial species of the genus Anabaena occurred at most sampling points with huge salinity differences (0.1-32.5 psu). We isolated several clones of Anabaena spp. from the monitoring stations, and screen out two euryhaline and nitrogen-fixing Anabaena clones, CB-MAL21 and CB-MAL22. The two clones were grown under various environmental gradients such as temperature (20, 30, 35 and 40$^{\circ}C$), salinity (0, 2, 5, 15 and 30psu), and $PO_4^{3-}$-P concentration (0, 1.6, 8.0, 40 and 200 ${\mu}M$M). Growth of CB-MAL21 and CB-MAL22 was measured by daily monitoring of chlorophyll fluorescence from each experimental culture for more than three serial transfers. Both the two experimental clones did not grow at 0psu. Maximal growth rates of the two clones were markedly reduced at lower $PO_4^{3-}$-P concentrations showing negligible growth at 0 and 1.6 ${\mu}M$M. However, growth of CB-MAL21 was not affected by low $NO_3^--$ concentration in culture media, showing the nitrogen-fixing ability. Maximum biomass yields of the two clones decreased dramatically at 35 and 40$^{\circ}C$. Optimal growth conditions for the two experimental clones were determined to be 20-30$^{\circ}C$, 40 ${\mu}M$M $PO_4^{3-}$-P, and wide salinity range from 5.0 to over 30psu. Best growth of CB-MAL21 was shown at (20$^{\circ}C$-15psu), which is less saline and cooler condition than those (i.e., 30$^{\circ}C$-30psu) for the best growth of CB-MAL22. The euryhaline and nitrogen-fixing CB-MAL21 strain thus can be a candidate laboratory culture for the future cyan bacterial marine biotechnology in temperate coastal waters.

• Advances in nano research
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• 제11권6호
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• pp.667-678
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• 2021

Multiwalled carbon nanotubes (MWCNTs) were first oxidized (O-CNTs) to introduce carboxylic group and then further functionalized (F-CNTs) with m-phenylenediamine, which was confirmed by FTIR and SEM. It was used as an effective adsorbent for the adsorptive removal of diclofenac drug from water. Under optimum conditions of pH 6, stirring speed 600 rpm, the maximum adsorption capacity obtained was 532 mg g^-1 which is superior to the values reported in literature. The adsorption was quite rapid as 25 mg L^-1 drug solution was adsorbed in only 3 minutes of contact time with 10 mg of adsorbent dose. The adsorption kinetics and isotherms were studied using various models to evaluate the adsorption process. The results showed that the data best fit in kinetics pseudo-second order and Langmuir isotherm model. Furthermore, the oxidized and functionalized MWCNTs were applied on gram-negative Escherichia coli and gram-positive Staphylococcus aureus using agar disc diffusion assay to validate their anti-microbial activity. Results were unique as both oxidized and functionalized MWCNTs were equally active against both E. coli and S. aureus. The newly synthesized F-CNTs have great potential in water treatment, with their dual action of removing drug and pathogens from water, makes it potential applicant to save environment.

• Applied Biological Chemistry
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• 제38권2호
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• pp.111-117
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• 1995

CRP (cAMP receptor protein)은 cAMP와 결합하여 cAMP-CRP 복합체를 형성하여 전사조절의 조절인자로서 작용한다. crp 유전자에 변이를 도입하여 cAMP의 비존재 상태에서 cAMP-CRP와 비슷한 기능을 가진 crp 유전자가 도입된 대장균 MK2001 (crp, cya::km)을 숙주로 사용하여 cAMP 혹은 cGMP의 비존재하에서도 mal 유전자의 발현을 촉진시키는 유전자 sfs (sugar fermentation stimulation) 수 종을 클로닝 하였다. 본 실험에서는 이미 밝혀진 nlp (Ner like protein) 유전자와 같이, sfs의 새로운 유전자를 탐색하여, 그 중 sfs4의 2126 bp 전 염기배열을 결정하고, 잠정적인 sfs4의 promoter 영역에는 CRP 단백질과의 결합 DNA 공통 염기배열(5' AAT TGTGA ACACCA TCACC CGT 3')이 존재함을 확인했다. lacZ 융합 유전자를 작성하여 TP2010R1 MK2001의 균주에서 cAMP를 첨가할 경우 각각 2.3배, 1.8배의 ${\beta}-galactosidase$ 활성이 증가하는 것으로 보아 sfs4는 cAMP-CRP에 의해 발현 조절을 받는 것으로 나타났다.

• Bulletin of the Korean Chemical Society
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• 제33권9호
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• pp.3091-3094
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• 2012