• Archives of Pharmacal Research
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• 제20권1호
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• pp.46-52
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• 1997

To obtain more sensitive immunoassay for methamphetamine (MA) determination, the optimum condition of enzyme-linked immunosorbent assay (ELISA) was investigated in regard to immunogens, antibody purification methods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetamine (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were purified by protein G chromatography or various immunoaffinity chromatography-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumin (OVA). Each purified antibody was characterized by means of sensitivity and cross-reactivity using the three MA-protein coating tracers, MA-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was acquired with the MA-OVA tracer although the tracer concentration and the antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimum condition, immunoaffinity chromatography-purified antibodies were better for sensitivity and for specificity than protein G-purified antibodies. The cross-reactivity of the purified antibodies seemed to be affected by immunogen structure and showed somewhat different patterns according to the immunoaffinity ligand utilized. These data show that the antibody purification method as well as choice of coating tracer and immunogen is essential for the sensitivity and specificity of EIA; the optimum condition for assay should be discovered using various methods and combinations.

• KSBB Journal
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• 제25권3호
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• pp.257-264
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• 2010

Bone morphogenetic protein-7 (BMP-7) is one of important growth factors for skeletal development and bone growth. In this work, BMP-7 was efficiently expressed in recombinant Bacillus subtilis. The mature BMP-7 protein indicated molecular weight of 15.4 kDa by Western blot assay and was secreted into culture medium with 0.35 ng/mL. The extracellular and intracellular rhBMP-7 proteins were purified by using a FPLC system with an ion exchange column and a gel filtration column. The extracellular and intracellular rhBMP-7 proteins had finally a 57.1% purity and a 36.2% purity, respectively. The purified rhBMP-7 proteins showed an intact biological activity which stimulated alkaline phophatase (ALP) activity in MC3T3-E1 cells.

• 미생물학회지
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• 제32권4호
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• pp.297-300
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• 1994

토양에서 분리한 방선균에서 제한효소 활성을 조사하여 그 가운데 Streptomyces diastatochromogenes로부터 제한효소 활성을 확인하였다. 이 균주의 제한효소 SdiI을 균체 15g으로부터 streptomycin sulfate침전, ammonium sulfate 침전, hydroxylapatite colume chromatography, Sephacryl S-200 HR column chromatography, hydroxylapatite column chromatography의 단계를 거쳐 분리하였고, SDS-polyacrylamide gel-electrophoresis에 의하면 소단위체 분자량은 약 35,000Da으로 추정되었다.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1994년도 한국생물과학협회 학술발표대회
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• pp.185.2-185
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• 1994

No Abstract, See Full Text

• 한국동물학회:학술대회논문집
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• 한국동물학회 1997년도 한국생물과학협회 학술발표대회
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• pp.270.1-270
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• 1997

No Abstract, See Full Text

• 한국환경과학회지
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• 제7권6호
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• pp.853-857
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• 1998

Purification of the isocitrate lyase extracted from Microbacterium laevaniformans was investigated. The isocitrate lyase was purified 43.6 folds by the following continuous treatment with ammonium sulfate fraction, DEAE-cellulose, DEAE-sephacel and Sephadex G-200 chromatography. The purified isocitrate lyase was showed to be a single protein band by polyacrylamide gel electrophoresis. The molecular weight of the purified isocitrate lyase was estimated 54,000 Da by the SDS-polyacrylamide gel electrophoresis. The Km and Vmax values for isocitrate were estimated to be 0.83mM and 0.33units/ml, respectively. Activity of isocitrate lyase was inhibited by cystein-HCl and glutathione.

• BMB Reports
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• 제49권11호
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• pp.585-586
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• 2016

Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.540-540
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• 2003

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권2호
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• pp.82-86
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• 1998

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins in E. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated buy centrifugation, resulting in highly purified proteins in the supernatant.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.881-884
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• 1999

The gene encoding the RNA-dependent RNA polymerase of the hepatitis C virus was cloned and expressed with a C-terminal hexahistidine tag. The protein was purified from Escherichia coli to near homogeneity and characterized in vitro. When the 21 amino acids from the C-terminus of the protein were deleted, an inclusion body was not formed and a better purification yield was achieved. However, the activity of the purified enzyme decreased compared to that of the full length protein. The purified enzyme did exhibit ribonucleotide-incorporation activity on an in vitro transcribed RNA containing the 3' end of the HCV genome. It also possessed ribonucleotide incorporation activity, to a lesser extent, on in vitro transcribed foreign RNA templates when RNA or DNA primers were present. The activity was higher with DNA primers than with RNA primers. Accordingly, this assay system will facilitate the screening of inhibitors for hepatitis C virus replication.