• Journal of Microbiology and Biotechnology
• /
• 제17권5호
• /
• pp.728-732
• /
• 2007

In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT(GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A(MT2A). There are in-framed multiple cloning sites(MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame(ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatography, known as $Ni^{2+}$-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step $Ni^{2+}$-affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30mg/l and 28mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins.

• Journal of Microbiology and Biotechnology
• /
• 제6권4호
• /
• pp.286-291
• /
• 1996

A study was conducted to investigate the characteristics of segregation and alcohol fermentation of intergeneric fusants. The protoplast fusion of both Pichia stipitis CBS 5776 and Saccharomycess cerevisiae STV 89 was carried out. The fusion frequency was $5\times10^{-8}$ and among fusants selected, a fusant F5 showed the best results in ethanol production by sucrose and xylose fermentations. The performance of xylose fermentation by this fusant was better than that of P. stipitis CBS 5776 and fusant F5 exhibited sucrose fermentation patterns intermediate to the two parent strains. The fusant F5 was segregated into a pair of parental strains during the several culture passages. In the average, 91$%$ of colonies had a similar characteristics of P. stipitis while 7$%$ of colonies resembled S. cerevisiae. Only 2$%$ of colonies had the characteristics of the original fusants. At the sixth passage, all segregants resembled P. stipitis. From these results it is suggested that intergeneric protoplast fusion led to an integration of S. cerevisiae genes, rather than whole chromosomes, within the entire genome of P. stipitis.

• Journal of Microbiology
• /
• 제33권2호
• /
• pp.120-125
• /
• 1995

.betha.-galactosidase activity of Escherichia coli cells containing operon fusion nhaA'-'lacZ was monitored to study the regulation of expression of nhaA gene under various conditions. The expression of the fusion was enhanced only by chemicals containing Na$^{+}$ or Li$^{+}$. This Na$^{+}$ or Li$^{+}$. This Na$^{+}$(Li$^{+}$)-specific enhancement of .betha.-galactosidase activity represented the increase in the rate of synthesis of .betha.-galactosidase rather than the decrease in the breakdown rate. The induction pattern was influenced by copy numbers of the gene. Induction by Na$^{+}$ or Li$^{+}$ was concentration and time dependent, reaching maximum 5-6 fold induction after 2 hours at 0.4-0.5 M for Na$^{+}$ or at 0.25-0.35 M for Li$^{+}$, Although the expression was induced at much lower concentration of Na$^{+}$ at alkaline pH values than at neutral pH in the presence of Na$^{+}$, alkaline pH itself did ot induced the expression of the fusion in the absence of Na$^{+}$. Temperature shift and growth phase of culture did not affect the level of induction.he level of induction.

• 미생물학회지
• /
• 제22권1호
• /
• pp.35-40
• /
• 1984

Streptomyces속 균주개발의 수단으로서 이용할 목적으로 원형질체 융합방법의 확립을 시도하였다. 특히 융합빈도를 높이고 실험을 간편화하는데 역점을 두었다. 원형질체의 형성 및 재생빈도는 균의 배양시간에 따라 변하였는데 대수기에서 수확한 균체로부터 가장 높은 빈도의 수율을 얻었다. 원형질체의 형성은 다른 용균효소를 사용하지 않고 Lysozyme 단독처리 만으로도 충분히 가능하였고 원형질체의 세포막 재생은 Monolay법 보다는 Overlay법이 훨씬 좋은 결과를 주었다. Monolay법은 1.8%, Overly법은 14%의 재생빈도를 나타냈다. 본 실험에서 PEG1000 (50% W/V)를 사용한 원형질체 융합방법으로 얻은 S. coelicolor의 재조합체의 빈도는 $1.8 {\times} 10^{-2}$이었다.

• BMB Reports
• /
• 제44권4호
• /
• pp.279-284
• /
• 2011

The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.

• Journal of Microbiology and Biotechnology
• /
• 제9권3호
• /
• pp.303-310
• /
• 1999

A cost-effective mass production method for a strong antimicrobial peptide, buforin II, which was isolated from the stomach of Bufo bufo gargarizans, has been developed. This method is based on the neutralization of the positive charge of buforin II by fusion with a cysteine-rich acidic peptide (CAP) to avoid any lethal effect on the host. The neutralized fusion peptide was multimerized and expressed in Escherichia coli as tandem repeats to increase the production yield. Multimers of the CAP-buforin II fusion peptide were successfully expressed at high levels in E. coli as inclusion bodies. More than 100mg of pure buforin II was obtained per 11 of E. coli culture after cleaving the multimeric polypeptide with CNBr. The buforin II obtained from the recombinant E. coli had antimicrobial activity identical to that of natural buforin II. The proposed expression system can provide a cost-effective mass production method for both antimicrobial peptides and other host-lethal basic proteins.

• 한국수정란이식학회지
• /
• 제19권3호
• /
• pp.191-199
• /
• 2004

본 연구는 돼지 태아 섬유아세포유래 공여세포를 미세주입에 의해 주입 후 재 조합한 핵 이식 배에 대한 배양액, 세포주기의 동기화, 배양시간 및 난자의 활성화에 따른 융합율과 체외발생율에 대해 조사하였다. 핵 이식 배를 NCSU-23, TL Hepes 및 TZM-3 배양액으로 1시간 및 8시간 배양하였을 때 배반포로의 분할율은 각각 15.6%, 14.0%, 15.0% 및 13.9%, 10.5%, 13.3%로서 배양액 및 시간에 따른 분할율의 유의적인 차이는 없었다. 공여핵원용 세포를 0, 8, 15시간 배양했을 때 G2/M기로의 체외발달율은 12.0%, 18.0%, 48.0%였다(p<0.01). 공여핵원용 세포를 12-24시간 배양했을 때 G2/M기로의 체외발달율은 유의한 증가를 나타내지 않았다. 공여핵원용 세포를 10% FBS + NCSU-23 배양액으로 1-2, 6-8, 12-14일간 배양 후 핵 이식한 배의 융합율은 각각 60.0%, 73.3%, 62.5%였으며, 분할율은 각각 36.0%, 56.7%, 50.0%였다. 0.5% FBS + NCSU-23, 0.5% + TL-Heaps 및 0.5% + TZM-3 배양액으로 5% $O_2$조건 하에서 배양하였을 때 핵 이식배의 $\geq$2 cell 및 배반포로의 발생율은 각각 12.5$\pm$1.6%, 11.1$\pm$1.8%, 11.7$\pm$1.0%였으며, 10% $O_2$조건 하에서 배양하였을 때 핵 이식배의 $\geq$2 cell 및 배반포로의 발생율은 각각 10.5$\pm$1.5%, 9.8$\pm$1.4%, 10.0$\pm$0.8%였다 배양액과 $O_2$ 조건에 따른 유의한 발생율에 차이는 인정되지 않았다.

• 한국균학회지
• /
• 제12권4호
• /
• pp.164-169
• /
• 1984

식용버섯의 재배와 소비가 증가해가는 중요성에 비추어 새로운 품종 육성을 위하여 국내의 주요 재배 버섯인 느타리버섯과 사철느타리버섯의 원형질체를 융합하여 종간 체세포 잡종을 선발하였으며, 그 특성을 조사한 결과를 요약하면 다음과 같다. 1. 느타리버섯과 사철느타리버섯의 종간 원형질체 융합율은 0.18-3.80%였다. 2. 융합된 균총의 생장은 빠른 것, 중간 그리고 아주 느린 것으로 나눌 수 있었으며 그 형태는 불규칙 하였다. 3. 선발한 40개의 체세포 잡종 모두가 정상적인 자실체를 맺었으며, 자실체의 형태가 모본과는 달리 아주 다양하게 나타났다. 4. 체세포 잡종의 자실체색은 짙은 회색을 가진 느타리버섯과 연한 백색을 가진 사철느타리버섯의 중간색을 나타내었다. 5. 무포자 또는 극히 적은량의 포자를 지닌 균주를 선발하였으며 이들의 자실체 형태는 정상이었다. 6. 수량이 다소 낮은 무포자 균주를 제외하고는 대체적으로 체세포 잡종의 자실체 수량이 모본보다 증가했다.

• 한국조리학회지
• /
• 제9권3호
• /
• pp.88-101
• /
• 2003

There is difference between European Cuisine and Korean Cuisine in various ways. In order to advance Korean Food into European Market, we should understand well European food-culture and food-custom. There are several process for push into the European Market. First, we inform European like that Korea traditional food (repast tool, the interior of Korean restaurant, traditional music, food-sequence, service method etc.). Second, we can introduce them ' Korean Fusion Food ' and that Korean Cuisine is ' Health foods '. For globalization of Korean Cuisine, we must understand exactly European market situation and know well the field law and tax system for business. Next, we establish the prior plan to supply with Korean food material till faraway region.

• 한국실내디자인학회:학술대회논문집
• /
• 한국실내디자인학회 2001년도 춘계학술발표대회 논문집
• /
• pp.86-89
• /
• 2001

Popular in the 1990s, Western Minimalism declined in popularity in the end of the century while the Oriental Mysticism and the Oriental Minimalism called “Zen Style” appeared. Fusion and Oriental Minimalism, both of which pursue traditional beauty and the essence of functionality and beauty, are combined with Japanese elements. As Oriental Minimalism is oriented towards Japan and China, we should consider the Japanese modernization process. Japan internationalized their own traditional elements after modernization. On the other hand, we only focus on tradition in a view of modernity, Japan developed their own culture whereas we followed the Western influences and trends after our culture and social structure were destroyed because of the civil war. Therefore, the aim of this paper is to find out the possibility of culture variety in interior design from the Japanese samples which modernized their culture in various sights. In other words, I researched and analyzed the expressive trends in Japanese Contemporary interior design, architecture and similarity and difference between Korea and Japan.