• Biotechnology and Bioprocess Engineering:BBE
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• 제5권1호
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• pp.13-16
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• 2000

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed in Escberichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced in E. coli cytoplasm were easily dissolved by simple alkaline pH shift $(8\rightarrow12\rightarrow8)$. Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.

• Preventive Nutrition and Food Science
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• 제1권1호
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• pp.106-110
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• 1996

Bromelain(EC 3.4.22.4), the collective name for the proteolytic enzymes found in tissues of the plant family Bromeliaceae(pineapple), has been used as a tenderizing agent in food processing, and as an antiinflammatory agent in pharmaceuticals. In this paper, we describe the simple purification method of bromelain using Korean pineapple fruit. After removing contaminants at 30% saturation of ammonium sulfate, the supernatant obtained was treated again with ammonium sulfate to 80% saturation. Wit the above salt fractionation, partially purified bromelain could be obtained. To get highly purified bromelain, the previous 30% to 80% ammonium sulfate treated precipitate was dialyzed against 25mM sodium acetate buffer(pH 5.0) followed by passing through a CM- cellulose cation exchange column. Fruit bromelain was eluted as a major peak at 0.5~0.8M NaCI gradient. The present method is simpler with high wield than the traditional purification method-acetone treatment and several consecutive chromatographic processes.

• Transactions on Electrical and Electronic Materials
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• 제9권5호
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• pp.209-212
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• 2008

We developed a simple and effective purification method to obtain high-purity single-walled carbon nanotubes (SWCNTs) with low surface damage. The purification process consists of oxidization at $430^{\circ}C$ for 1 h in a furnace system of air atmosphere and homogenization in dilute hydrochloric acid solution for extremely short time. The role of homogenizer was examined during purification process in terms of purity and quality of purified SWCNTs. High-purity and low surface damage of SWCNT products was obtained using homogenizer which was operated at 8500 rpm for 10 min in the environment of 7 % HCI solution. From XRD spectra, we observed that metal catalysts were thoroughly removed. Raman spectra showed that the intensity values of crystallization ($I_{G}/I_{D}$) of purified SWCNTs were very similar with that of pristine SWCNTs. Moreover, the structure damage of purified SWCNTs was hard to find from electron microscopy. Consequently, homogenizing, which is a quick and simple manner, can be promising method for obtaining final SWCNTs with clearly high purity and crystallinity.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.521-524
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• 2003

An effective purification method was developed for producing Homoharringtonine (HHT), to guarantee high purity and yield from Cephalotaxus koreana. This process was a simple and efficient procedure, for the isolation and purification of HHT form the biomass of Cephalotaxus koreana, consisting of extract, adsorbent treatment, precipitation and followed by a chromatography. The extraction, adsorbent treatment and precipitation in pre-purification process allows for rapid and efficient separation of HHT from many compound and dramatically increases the yield and purity of crude HHT for HPLC purification steps compared to alternative processes. This purification processes serves to minimize solvent usage, size, and complexity of the operations for HHT purification.

• Journal of Microbiology and Biotechnology
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• 제19권7호
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• 약학회지
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• 제47권6호
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.986-988
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• 2002

A plasmid expression vector of pEStxl encoding a mature form of the B chain of the Shiga toxin was constructed without a signal peptide under the control of an inducible n promoter. The encoded protein was purified to 90% by simple heat treatment, and then further purified to 95% by Phenyl-Sepharose and DEAE-Sepharose chromatographies, all in a single day. Accordingly, this expression system and heat treatment could facilitate the rapid purification of gram-scale amounts of the Shiga toxin B subunit from recombinant Escherichia coli cells.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권6호
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• pp.423-427
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• 2004

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of $80.1\%$ by ammonium sulfate precipitation and a single chromatographic step involving FPLCanion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from $21.5{\~}29$ kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.

• KSBB Journal
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• 제15권4호
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• pp.342-345
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• 2000

식물세포배양으로부터 peroxidase를 대량 생산하기 위한 분리/정제 공정 으로서 세포를 파쇄하고 크로마토그래피 전처리로서 활성백토를 적용하였다. 활성백토는 미세한 세 포 조각 뿐만 아니라 여려가지 불순불을 선택적으로 흡착 하는 성질을 나타내었으며, 이를 통하여 효과적으로 정제 공정을 진행할 수 있었다 흡착을 통한 전처리 후 한외여 과장치를 이용하여 농축을 실시 하였으며, DEAE-Sepharose F FF를 이용한 크로마토그래피를 통해 정제가 이루어졌다. 활성을 나타내는 용액을 탈염, 농축 후 동결 건조하였다. 이 공정은 상업화에 필요한 대량 정제를 가능하게 하였으며 수율과 정제비용 측면에서 상당히 경제적인 공정임을 입증하였다, 활성백토를 이용한 흡착공정은 다른 효소 및 단백질의 경제적인 대량정제에 적용될 수 있으리라 기대된다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.566-569
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• 2005

한국산 개비자나무로부터 새로운 개념의 homoharringtonine 분리 및 정제 공정을 개발하였다. 메탄올을 사용한 4회 반복 추출에 의해 biomass로부터 대부분(>99%)의 homoharringtonine을 회수 할 수 있었다. 흡착 공정에서는 활성 백토(active clay)를 사용하여 추출물에 포함되어 있는 식물유래 타르, 왁스 성분을 효과적으로 제거하였다. Silica gel low-pressure chromatography공정을 통해서 순도 52% 이상의 homoharringtonine을 얻었으며, HPLC 공정을 통해 고순도 및 고수율의 homharringtonine을 정제할 수 있었다. 정제된 homoharringtonine의 분자량 및 구조분석을 수행한 결과 표준물질(Homharringtonine)과 동일 물질임을 확인할 수 있었다.