• 한국미생물·생명공학회지
• /
• 제15권6호
• /
• pp.396-401
• /
• 1987

$\beta$-Galactosidase 유전자 조작된 두 대장균 숙주-벡터 시스템의 단백질 생산가 배양특성을 비교하였다. Tac 프로모터를 갖는 Escherichia coli JM109/ pTBG10 균주가 pL 프로모터를 갖는 E. coli MH 3000(pRKcI857)/ pASl(lacz) 균주보다도 단백질 생산성에 있어서 훨씬 우수하였으며 초기 및 중간 대수 증식기에서의 발현 유도가 단백질 생산에 있어서 유리함을 관찰하였다. 또한 유전자 발현시기에 있어서는 산소요구량이 매우 높았으나 pH를 일정하게 조절함에 의해 산소요구량을 어느 정도 낮출 수 있었고 생산성도 향상됨을 관찰할 수 있었다. 고생산성 균주(E. coli JM109/pTBG10)의 경우 플라스미드 함유 균체의 플라스미드 상실 균체에 대한 비증식속도의 비 $\mu$ + /$\mu$- 가 저생산성 균주보다도 낮았으며 이것은 유전자발현에 따른 단백질 합성부담이 매우 큰 때문으로 판단되었다. 또 플라스미드 안정성을 30세대까지 추적하였는바 유전자 발현 조건하에서 두 균주 모두 25세대 후 10% 정도의 안정성을 보였으나 E. coli JM109/pTBG10 균주의 안정성이 상대적으로 우수하다고 판단되었다.

• Journal of Microbiology and Biotechnology
• /
• 제25권11호
• /
• pp.1835-1841
• /
• 2015

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

• 대한수의학회지
• /
• 제39권4호
• /
• pp.786-796
• /
• 1999

As an attempt to develop an effective control method against theileriosis, recombinant antigen protein was produced. Thirty-two kDa membrane protein(MP) gene of T sergenti was amplified through RT-PCR from extracted total RNA of T sergenti isolated in Chonbuk, Korea. The amplified 869 bp of Korean T sergenti membrane gene was cloned and the base sequences were analyzed. The amplified gene was cloned into E coli expression vector, pQE32 plasmid vector, and the vector was introduced into E coli strain M15 to produce the recombinant membrane protein. For the induction of T sergenti membrane protein(KTs-MP), the plasmid harboring E coli strain M15 were cultured in the presence of IPTG, and the recombinant protein were purified by $Ni^+$-NTA agarose. Then, to confirm the authenticity of the produced membrane protein, molecular weight of expressed recombinant KTs-MP was analyzed by SDS-PAGE and Western blotting. The molecular weight of expressed recombinant protein was 32 kDa as expected. The recombinant KTs-MP was successfully recognized by anti-His Tag antibody, antisera of T sergenti infected cattle and monoclonal antibody of T sergenti membrane protein. Therefore, we concluded that the authentic 32 kDa membrane protein of T sergenti was produced as immunologically recognizable form.

• KSBB Journal
• /
• 제24권4호
• /
• pp.341-346
• /
• 2009

본 연구에서는 Candida antarctica로부터 genomic DNA을 추출하여 lipase A(CalA) 유전자를 PCR 증폭하였고, 재조합 pColdIII/CalA, $pPICZ{\alpha}A$/CalA, $pPICZ{\alpha}A$/CalA$his{\times}6$을 구축하였다. 재조합 CalA 유전자의 기능적 발현을 위해 최적화된 시스템을 구축하고자 Escherichia coli와 Pichia pastoris 시스템에서 각각 수행하여 비교, 분석하였다. SDS PAGE gel을 통해 CalA의 발현의 여부 및 발현양을 확인하였고, pNPP를 기질로 한 가수분해 반응을 통해 활성을 측정하였다. E. coli 발현 시스템은 형질전환 방법이 간단하고, 미생물의 성장 속도가 빠르다는 장점을 갖지만 CalA의 활성이 0.02 Unit/ml으로 비교적 낮았으며 세포질 (cytoplasm)에서 발현되므로 비목적 단백질과의 분리 및 정제과정이 필요하다. 재조합 $pPICZ{\alpha}A$/CalA을 P. pastoris 시스템에서 발현한 경우 높은 발현양 뿐만 아니라 분비작용으로 인해 고순도 발현이 용이하였고, 활성 또한 약 0.7 Unit/ml으로 가장 높았다. 결론적으로 CalA의 기능적 발현을 위해 P. pastoris 시스템을 구축하는 것이 가장 적합함을 확인하였다.

• 대한수의학회지
• /
• 제45권2호
• /
• pp.185-189
• /
• 2005

Actinobacillus pleuropneumoniae is the causative agent of a porcine contagious pleuropneumonia. Among several virulence factors including exotoxin (Apx toxins), LPS, transferrin-binding proteins, OMPs, and some proteases, Apx toxins have been major targets for the protection study. In this study, cloning and expression of A. pleuropneumoniae Apx I and Apx II toxin, which are produced by all highly virulent strains, were performed by Escherichia coli expression system. Genes coding Apx I and II toxin were amplified from the A. pleuropneumoniae serotype 5 genomic DNA using polymerase chain reaction and cloned to a prokaryotic expression vector, pRSET. Expression of the Apx I and Apx II coding sequences in E. coli resulted in the formation of insoluble inclusion bodies purified according to a denaturing purification protocol, which employs the use of guanidium. Recombinant proteins were purified using $Ni^{2+}$-charged resin affinity purification. This expression and purification system made it possible to produce Apx I and Apx II in large amounts for further immunologic studies.

• Journal of Microbiology and Biotechnology
• /
• 제24권9호
• /
• pp.1232-1237
• /
• 2014

Lycopene, which is a well-known red carotenoid pigment, has been drawing scientific interest because of its potential biological functions. The current study reports that lycopene acts as a bactericidal agent by inducing reactive oxygen species (ROS)-mediated DNA damage in Escherichia coli. Lycopene treatment elevated the level of ROS-in particular, hydroxyl radicals ($^*OH$)-which can damage DNA in E. coli. Lycopene-induced DNA damage in bacteria was confirmed and we also observed cell filamentation caused by cell division arrest, an indirect marker of the DNA damage repair system, in lycopene-treated E. coli. Increased RecA expression was observed, indicating activation of the DNA repair system (SOS response). To summarize, lycopene exerts its antibacterial effects by inducing $^*OH$-mediated DNA damage that cannot be ameliorated by the SOS response. Lycopene may be a clinically useful adjuvant for current antimicrobial therapies.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 1991년도 춘계학술발표대회 논문집
• /
• pp.237-242
• /
• 1991

In order to increase the production of glutathione by maximizing the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were cloned. A gshI gene was cloned onto pBR322 plasmid as 3.6Kb PstI DNA fragment from E. coli K-12 chromosomal DNA. Also gshII gene was cloned onto pUC13 plasmid as 2.2Kb PstI-BamHI DNA fragment. In order to improve the glutathione producing activity more efficiently, various recombinant plasmids containing tandem repeated gshI genes or both genes in various copy number onto the same vector were constructed. E. coli cells harboring pGH501 plasmid (pUC8-gshI$\cdot$I$\cdot$II) showed the highest glutathione synthesizing activity. The conditions for glutathione production with an ATP-generating system such as acetate kinase reaction of E. coli cells or glycolytic pathway of yeast cells were examined using the E. coli cells harboring the pGH501 plasmid. When the acetate kinase reaction of E. coli cells was used as an ATP generating system, 20mM of L-csteine was converted into glutathione with a yield of $100\%$.

• 미생물과산업
• /
• 제19권4호
• /
• pp.14-26
• /
• 1993

Optimizing protein production in recombinant E. coli strains involves manipulation of genetic and environmental factors. In designing a production system, attention must be paid to gene expression efficiency, culture conditions and bioreactor configuration. Although not much emphasis was given to the physiology of host strains in this review, an understanding of the relationship between the physiology of host cell growth and the overproduction of a cloned gene protein is of primary importance to the improvement of the recombinant fermentation processes. Sometimes it is desirable to make use of gene fusion systems, e.g. protein A, polypeptide, gutathione-S-transferase, or pneumococcal murein hydrolase fusion, to facilitate protein purification.

• Journal of Microbiology and Biotechnology
• /
• 제23권3호
• /
• pp.322-328
• /
• 2013

In this study, we show the expression and purification of the human recombinant farnesoid X receptor (FXR)- ligand binding domain (LBD) protein in E. coli using a double cistronic vector, pACYCDuet-1, as a soluble form. We describe here the expression and characterization of a biologically active $FXR-LBD_{(248-476)}$. When expressed in the influence of bacterial promoters ($P_{T7}$ and $P_{Tac}$) of the single cistronic expression vectors, the human recombinant $FXR-LBD_{(248-476)}$ was found to be totally insoluble. However, by using a double cistronic expression vector, we were able to obtain the human recombinant $FXR-LBD_{(248-476)}$ in a soluble form. To allow for biological activities, we have subcloned into the pACYCDuet-1 vector, expressed in E. coli cells at some optimized conditions, and purified and characterized the human recombinant active $FXR-LBD_{(248-476)}$ proteins using the fluorescence polarization assay. This suggests that the expression of FXR-LBD in a double cistronic vector improves its solubility and probably assists its correct folding for the biologically active form of the proteins. We suggest that this may represent a new approach to high expression of other nuclear receptors and may be useful as well for other classes of heterodimeric protein partners.

• Journal of Microbiology and Biotechnology
• /
• 제30권3호
• /
• pp.333-340
• /
• 2020

Macrophages are the cells of the first-line defense system, which protect the body from foreign invaders such as bacteria. However, Gram-negative bacteria have always been the major challenge for macrophages due to the presence of lipopolysaccharides on their outer cell membrane. In the present study, we evaluated the effect of phloretin, a flavonoid commonly found in apple, on the protection of macrophages from Escherichia coli infection. RAW 264.7 cells infected with standard E. coli, or virulent E. coli K1 strain were treated with phloretin in a dose-dependent manner to examine its efficacy in protection of macrophages. Our results revealed that phloretin treatment reduced the production of nitric oxide (NO) and generation of reactive oxygen species along with reducing the secretion of proinflammatory cytokines induced by the E. coli and E. coli K1 strains in a concentration-dependent manner. Additionally, treatment of phloretin downregulated the expression of E. coli-induced major inflammatory markers i.e. cyclooxygenase-2 (COX-2) and hemeoxygenase-1 (HO-1), in a concentration dependent manner. Moreover, the TLR4-mediated NF-κB pathway was activated in E. coli-infected macrophages but was potentially downregulated by phloretin at the transcriptional and translational levels. Collectively, our data suggest that phloretin treatment protects macrophages from infection of virulent E. coli K1 strain by downregulating the TLR4-mediated signaling pathway and inhibiting NO and cytokine production, eventually protecting macrophages from E. coli-induced inflammation.