• Journal of Microbiology and Biotechnology
• /
• 제15권5호
• /
• pp.1022-1027
• /
• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• 유통과학연구
• /
• 제15권4호
• /
• pp.15-23
• /
• 2017

Purpose - This research aims to determine whether the consumer affinity and ethnocentrism as well as the image of a foreign country (Japanese product as the most popular product in Indonesia) are able to influence behavior related to the perceived risk and willingness to buy foreign products from the affinity country. Research design, data, and methodology - Using survey techniques with 164 respondents, the study uses structural equation model with confirmatory factor analysis (CFA). To ensure the research objective and appropriate respondent, then we select an individual who have interest on Japanese culture & language. The primary and secondary data used in this study. Primary data refers to information collected directly from respondent by questionnaires dissemination while secondary data is provided from well-established literatures. Results - The results show us that the ethnocentrism has dominant affection role compared to affinity in order to influence consumer behavior meanwhile, the product country image has cognition role to evoke consumer desire to consume foreign products. Conclusions - From a theoretical perspective, the study contributes to international marketing literature by refining the conceptualization of the consumer affinity construct and highlighting its multidimensional nature. The consumer affinity research need to enrich in term of the context and the different culture and situation.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
• /
• pp.703-706
• /
• 2000

We constructed a biospecific affinity surface using hyper-branched dendrimers on gold for biospecific recognition, and characterized the resulting surfaces by using confocal fluorescence microscopy. The dendrimer monolayer was firstly constructed on the mercaptoundecanoic acid SAM/Au with pentafluorophenyl ester activation and further functionalized with sulfo-NHS-biotin, an activated ester of biotin. To confirm the formation of biospecific affinity surface, FITC(fluorescein isothiocyanate)-labeled avidin was loaded onto the biotinylated dendrimer monolayer, and fluorescence images of the bound avidins were investigated with a confocal microscope. The constructed biospecific affinity surface showed a much more dense and uniform fluorescence compared to those from poly-L-lysine- and cystamine SAM-based affinity surfaces. For the dependency on the concentration of added FITC-labeled avidin on the affinity surface, derived fluorescence could be detectable from as low as $1{\mu}g/ml$, and intensified up to $50{\mu}g/ml$. Further reaction of FITC-labeled avidin layer with TMR(tetramethylrhodamine)-biocytins resulted in the efficient FRET(fluorescence resonance energy transfer) phenomenon. As an extension of the study, we attempted a patterning of the affinity surfaces on gold by microcontact printing. Fluorescence of the patterned surface demonstrated that FITC-labeled avidin molecules were specifically bound to the biotinylated patches.

• Journal of Plant Biology
• /
• 제30권4호
• /
• pp.277-285
• /
• 1987

Undifferentiated suspension cells had the ability to transfer glucose and fructose actively, but the suspension culture cells were unable to transfer saccharide without previously splitting to monosccarides. The uptake of fructose showed the low- and high-affinity system compared to of glucose, which possessed only one saturable uptake system. In this paper, the low affinity system of the uptake of fructose has been studied intensively. Glucose did not inhibit the low affinity system of fructose competitively. The Km value was 47 mM for fructose, 7.4 mM for glucose and Vmax was 69 $\mu$mol/h.g fresh weight for fuctose, 9.8 $\mu$ mol/h.g fresh weight for glucose. Metabolizer inhibitors, both 50 $\mu$M of CCCP and DNP, inhibited 70% of the uptake of the low affinity system of fructose. The proton ions were accompanied by the uptake of fructose. The stoichiometry showed ratio of proton to fructose was 0.17. The mechanism ofthe uptake was fructose-proton-symport. The molecules of fructose accmululated inside 25 times more than outside. Therefore, the low affinity system of fructose was not mere diffusion, but depended on metabolic energy and thus transported actively. The importance of this system was discussed.

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 1998년도 학술발표회
• /
• pp.29-29
• /
• 1998

Previously, we reported two types of vanadate-sensitive ATPases in the micro somes of tracheal epithelial cells, a high-affinity one and a low-affinity one. The low affinity vanadate-sensitive (LAVS) ATPase was sensitive to thapsigargin and cyclopiazonic acid, specific antagonists of ER-type Ca$\^$2＋/-ATPase, and mediated microsomal $\^$45/Ca$\^$2+/ uptake, implying that the LAVS-ATPase is an ER/SR-type Ca$\^$2+/-ATPase.(omitted)

• Bulletin of the Korean Chemical Society
• /
• 제13권2호
• /
• pp.119-122
• /
• 1992

The affinity chromatographic technique was applied to the isolation of Thromboxane Synthase, with a variety of imidazolyl alkanoic acids coupled Sepharose 2B including a gel (G in Table 4) which has one free COOH group in the bound affinity ligand. The effect of ligand structure on the "affinity" and "selectivity" for thromboxane synthase isolation is described.

• 미생물학회지
• /
• 제21권4호
• /
• pp.221-228
• /
• 1983

Saccharomyces cerevisiae로 부터 glucose-6-phosphate dehydrogenase을 신속하고 간편하게 정제하는 과정을 affinity chromatography를 이용하여 개발하였다. 이 효소를 정제하는데 적절한 affinity medium을 조사해 본 결과, $NADP^+ -agarose$와 Affi-gel Blue(Cibacron Blue F3GA)가 Affi-gel Red(Procion Red HE-3B), AMP-agarose, ATP-agarose, 그리고 $NADP^+ -agarose$보다 유용함이 밝혀졌다. 이 두가지 affinity media에 흡착된 효소를 분리 하는데 가장 적합한 elution 조건을 조사하였는데 KCI gradient( (0-1.OM)가 효소의 순도 및 수회율을 가장 높일 수 있는 적합한 방법이었다. 특히 Affi-gel Blue를 사용할 경우, KCI gradient로 효소를 용출시키기 전에 NAD-(15mM)로NAD+에 친화역을 갖는 효소들을 제거하는 것이 enzyme의 순도를 높이는데 매우 효과적이었다. 그 결과 glucose-6-phosphate dehydrogenase를 baker's yeast로 부터 기존의 간단한 정제 과정과 affinity chromatography를 병행한 방식을 샤용하여 분리 하였는데, affinity medium으로 Affi-gel Blue를 사용했을 때는 180배 정도, NADP+-agarose를 사용했을 때는 2,000배 정도로 정제 되었다. 대량으로 glucose-6-phosphate dehydrogenase를 정제하는 경우, Affi-gel Blue를 사용하던 효소의 순도는 NADP+-agarose보다 낮으나, 효소의 회수율은 훨씬 더 높았다. 또한 G-6-P dehydrogenase에 대한 affinity medium의 capacity도 Affi-gel Blue가 NADP+-agarose보다 5배정도 높았으며 더우기 Affi-gel Blue는 여러번 반복적으로 사용될 수 있고, 그 제조 과정도 NADP+-agarose보다 간단하며 경비도 적게 들었다.

• 멤브레인
• /
• 제19권2호
• /
• pp.113-121
• /
• 2009

실리카 입자를 기공 형성제로 사용하여 물리적 강도와 단백질 결합용량이 높은 다공성 키토산 및 키틴 친화 막을 제조하였다. 키토산 친화 막의 BSA 단백질 결합용량은 최대 21.8mg/mL이었으며, 키틴 친화 막의 lysozyme 효소 결합용량은 최대 26.1mg/mL이었다. 제조된 다공성 키토산 및 키틴 친화 막을 사용하여 단백질 용액의 loading 유량, loading 양 및 농도 변화에 따른 BSA와 lysozyme의 친화 막 여과 크로마토그래피 분리 실험을 수행하였다. 친화 막 여과 크로마토그래피 분리 실험을 통해 얻어진 loading/washing/elution의 단계로 구성된 일련의 크로마토그램으로부터 단백질 용출량과 결합수율을 구하였다. 키토산 및 키틴 친화 막에의 BSA 및 lysozyme 단백질의 결합량과 결합수율은 loading용액의 유량이 작을수록, 주입량 및 농도가 클수록 증가하였다. 이 결과로부터 실리카 입자를 기공 형성제로 사용하여 제조된 다공성 키토산 및 키틴 막은 단백질의 대규모 여과 크로마토그래피 분리를 위한 친화 막으로서 효과적인 활용이 기대된다.

• BMB Reports
• /
• 제33권6호
• /
• pp.531-536
• /
• 2000

Tropomyosin (TM) is an important actin binding protein involved in regulation of muscle contraction. Unacetylated striated tropomyosin failed to bind to actin whereas unacetylated smooth tropomyosin bound well to actin. It has been demonstrated that high actin affinity of unacetylated ${\alpha}-tropomyosin$ was ascribed to the carboxyl terminal amino acid residues. In order to define the role of the carboxyl terminal residues of tropomyosin molecule on actin binding, two mutant tropomyosins were constructed. TM11 is identical to the striated tropomyosin except that the carboxyl terminal last three amino acids was replaced with $^{282}NNM^{284}$ whereas in TM14 $^{276}HA^{277}$ was substituted with smooth specific $^{276}QT^{277}$. TM11 and TM14 were overproduced in Escherichia coli and analyzed for actin affinity. The apparent binding constants (Kapp) of unacetylated tropomyosins were $2.2{\times}10^6M^{-1}$ for sm9, $1.03{\times}10^6M^{-1}$ for TM14, $0.19{\times}10^6M^{-1}$ for TM11, $>0.1{\times}10^6M^{-1}$ for striated, respectively. This result indicated that higher actin affinity of the unacetylated smooth tropomyosin was primarily attributed to the presence of QT residues in the smooth sequence. In case of the Ala-Ser (AS) dipeptide extension of the amino terminus of tropomyosin, Kapp were $21.1{\times}10^6M^{-1}$ for AS-sm9, $8.0{\times}10^6M^{-1}$ for AS-11, $4.7{\times}10^6M^{-1}$ for AS-14, $3.8{\times}10^6M^{-1}$ for AS-striated. AS-TM11 showed considerably higher actin affinity than AS-TM14, implying that interaction of Ala-Ser of the amino terminus with the carboxyl terminal residues. Since Kapp of AS-TM11 was significantly lower than that of AS-sm9, the presence of QT might be required for restoration of high actin affinity of the smooth ${\alpha}-tropomyosin$. These results suggested that the carboxyl terminal amino acid residues Glutamine275-Threonine276 are important for actin affinity of the recombinant smooth ${\alpha}-tropomyosin$, particularly of unacetylated smooth ${\alpha}-tropomyosin$.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제9권2호
• /
• pp.107-111
• /
• 2004

A comparative study was performed to evaluate the signal amplification strategies in electrochemical affinity sensing, which included the direct electron transfer and diffusible-group mediated electron transfer between label enzymes that were specifically bound to target proteins and chemically modified electrode surfaces. As a platform surface for affinity recognition reactions, a double functionalized poly(amidoamine) dendrimer monolayer that was modified with ferrocene and biotin groups was constructed on a gold surface. With the chemically modified electrode, a model affinity sensing with avidin was investigated. The advantages of adopting the diffusible-group mediated signaling strategy were demonstrated in terms of signal sensitivity and stability.