• Macromolecular Research
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• 제11권1호
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• pp.53-61
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• 2003

A molecular-thermodynamic model is developed for the salt-induced protein precipitation. The protein molecules interact through four intermolecular potentials. An equation of state is derived based on the statistical mechanical perturbation theory with the modified Chiew's equation for the fluid phase, Young's equation for the solid phase as the reference system and a perturbation based on the protein-protein effective two body potential. The equation of state provides an expression for the chemical potential of the protein. In a single protein system, the phase separation is represented by fluid-fluid equilibria. The precipitation behaviors are simulated with the partition coefficient at various salt concentrations and degree of pre-aggregation effect for the protein particles. In a binary protein system, we regard the system as a fluid-solid phase equilibrium. At equilibrium, we compute the reduced osmotic pressure-composition diagram in the diverse protein size difference and salt concentrations.

• Macromolecular Research
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• 제11권4호
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• pp.241-249
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• 2003

A molecular-thermodynamic model to describe the salt-induced protein precipitation is developed based on the perturbation theory. We employed the modified perturbed hard-sphere-chain (PHSC) equation of state for copolymer mixtures to take into account the pre-aggregation effect among protein particles. Hypothetical pressure-composition diagrams are computed with various size differences and salt concentrations. The precipitation behaviors are also studied for various types of pre-aggregation effect for the given systems.

• Korean Chemical Engineering Research
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• 제43권2호
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• pp.236-242
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• 2005

대표적인 단백질 물질인 bovine serum albumin(BSA), lysozyme, pepsin, casein 그리고 ovalbumin의 이성분계 단백질 혼합물의 미세 분리 막에서 보이는 막 오염 현상에 관한 실험을 고찰하였다. 트랙 에치법에 의해 제조된 $0.2{\mu}m$ 폴리카보네이트 미세 막을 이용하여 단백질 혼합물 용액의 조성과 종류가 여과유량감속에 미치는 영향에 대한 실험을 일정한 압력(14 kPa)과 수소이온농도(pH=11) 하에서 수행하였다. 각각의 이성분 단백질 혼합물에 대한 막오염 현상을 확인하였으며, 유량 감속 데이터는 최근에 개발된 pore blockage-cake formation 모델을 이용하여 분석한 결과, 실험 결과와 일치하였다. 이성분계 단백질 혼합물의 조성과 단백질 종류가 막오염 및 유속변화에 미치는 영향은 공극막힘속도(${\alpha}$), 단백질 침적층의 초기 저항($R_{po}$) 및 단백질 저항의 증가속도(${\beta}$)에 의해 설명 할 수 있었다. 대체로 공극막힘속도의 변화는 유속감소현상과 일치함을 알 수 있으며, 특히 casein이 존재하는 단백질 혼합물에서의 급격한 유량감속은 casein에 의한 단백질 침적층의 초기저항에 크게 기인된 것으로 판단된다.

• 한국식물병리학회지
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• 제11권1호
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• pp.66-72
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• 1995

The coat protein (CP) gene of cucumber mosaic virus-As (CMV-As) strain was engineered for expression in the plant by using the cauliflower mosaic virus 35S transcript regulatory sequences. The CP gene was cloned into an Agrobacterium-derived binary vector. A chimeric gene was constructed by the cDNA of CMV-As CP and plant expression vector pBI121. The clone, pCMAS66, was first introduced into the phagemid vector pSPORT1 for situating sense orientation for translation and making restriction sites in order to re-introduce plant expression vector, pHI121. The resulting subclone pCASCP02 and plant expression vector pBI121 were treated with BamHI-SacI for excising the target gene and removing GUS gene, respectively. After Agrobacterium transformation by freeze-thaw technique, the clone, pCMASCP121-123 which contains sense orientation of the target gene, was selected and confirmed by restriction endonuclease analysis. The CMV-As CP gene was introduced into A. tumefaciens. The results on tobacco plant transformation with the vector system revealed that the system could be successfully introduced and showed high frequency of selection to putative transformations.

• 대한환경공학회지
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• 제28권3호
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• pp.270-275
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• 2006

채취한 양식진주를 불순물 제거와 표면표백을 통해 염색이 용이하도록 전처리하였다. 전처리된 양식 진주의 표면은 해수로부터 형성된 경단백질의 일종인 Conchiolin 막으로 덮혀있기 때문에 Conchiolin 층에 흡착이 가능한 염료를 선택하여 상품으로 원하는 색상을 흡착하는 공정이 필요하다. Conchiolin 층에 흡착 가능한 염료들은 Rhodamine 6aG(R6G), Rhodamine B(RB) 및 Methylene Blue(MB) 등의 염기성 염료들이 주류를 이루고 있으며 이들 염기성 염료를 선택하여 각각 이성분 및 삼성분으로 염색용액을 제조하여 경쟁흡착 실험을 수행하였다. 이성분 및 삼성분 경쟁흡착의 친화도는 단일성분 흡착 model 인 Langmuir 혹은 Redlich-Peterson(RP) model과 결합된 ideal adsorbed solution theory(IAST)를 이용하였으며, 흡착자료와 IAST 예측치의 차이를 $R^2$ 및 SSE 값으로 판단하였다. 결과적으로 분급되지 않은 진주층에 대한 R6G와 RB의 경쟁흡착의 경우에는 IAST 예측치는 실험값과 잘 일치하고 있으나, 분말상 진주층의 경우, 높은 농도에서의 RB는 실험치와 예측치가 일치하고 있지 않음을 알 수 있었다. 분급된 진주층의 경우에도 R6G/RB, R6G/MB, MB/RB에서 이성분계 혼합용액의 이성분 경쟁흡착에서 R6G/RB의 경우에만 RB는 잘 일치하지 않음을 확인되었다. 삼성분계의 경우에도 RB를 제외하고는 실험치와 예측치가 잘 일치하고 있었다.

• BMB Reports
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• 제38권2호
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• pp.225-231
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• 2005

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus-as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.

• BMB Reports
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• 제35권2호
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• pp.186-193
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• 2002

The acid-bible subunit (ALS) associates with the insulinlike growth factor (IGF)-I or II, and the IGF binding protein-3 (IGFBP-3) in order to form a 150-kD complex in the circulation. This complex may regulate the serum IGFs by restricting them in the vascular system and promoting their endocrine actions. Little is known about how ALS binds to IGFBP3, which connects the IGFs to ALS. Xenopus oocyte was utilized to study the function of ALS in assembling IGFs into the ternary complexes. Xenopus oocyte was shown to correctly translate in vitro transcribed mRNAs of ALS and IGFBP3. IGFBP3 and ALS mRNAs were injected in a mixture, and their products were immunoprecipitated by antisera against ALS and IGFBP3. Contrary to traditional reports that ALS interacts only with IGF-bound IGFBP3, this study shows that ALS is capable of forming a binary complex with IGFBP3 in the absence of IGF When cross-linked by disuccinimidyl suberate, the band that represents the ALS-IGFBP3 complex was evident on the PAGE. IGFBP3 movement was monitored according to the distribution between the hemispheres. Following a localized translation in the vegetal hemisphere, IGFBP3 remained in the vegetal half in the presence of ALS. However, the mutant IGFBP3 freely diffused into the animal half, despite the presence of ALS, which is different from the wild type IGFBP3. This study, therefore, suggests that ALS may play an important role in sequestering IGFBP3 polypeptides via the intermolecular aggregation. Studies using this heterologous model will lead to a better understanding of the IGFBP3 and ALS that assemble into the ternary structure and circulate the IGF system.

• Molecules and Cells
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• 제43권11호
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• 한국해양바이오학회지
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• 제16권1호
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• pp.26-35
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• 2024

Chlorella has various biotechnological applications, including in the biomedical and pharmaceutical industries, because of its advantages, including rich nutrients, fast growth rate, easy cultivation, and high biomass. We used the Agrobacterium-mediated transformation method to express human GM-CSF and EGF proteins, which are widely used in regenerative medicine, cosmetics, and pharmaceutical materials in Chlorella. The codon-optimized hGM-CSF and hEGF genes were cloned into plant binary vectors and transformed into Chlorella vulgaris using the Agrobacterium-mediated coculture transformation method. After transformation, genomic DNA PCR was performed for each C. vulgaris line that was stably subcultured on an antibiotic-resistant solid medium to confirm the insertion of hGM-CSF and hEGF into the chromosome. Furthermore, PT-PCR and protein expression of hGM-CSF and hEGF in each transformed C. vulgaris were significantly increased compared to the untransformed Chlorella. This study suggests that high-value proteins, including hGM-CSF and hEGF, which are foreign genes of C. vulgaris, can be stably expressed through the Agrobacterium-mediated Chlorella transformation system.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.99-99
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• 2001

The acid-labile subunit (ALS) associates with insulin-like growth factor (IGF)-I or -II and IGF binding protein-3 (IGFBP-3) to form a 150-kD complex in the circulation. This complex is thought to regulate the serum IGFs by restricting them in the vascular system and promotes their endocrine actions. Little is known about how ALS binds to IGFBP3, which connects the IGFs to ALS. Xenopus oocyte was utilized to study the function of ALS in assembling IGFs into the ternary complexes. Xenopus oocyte was shown to correctly translate in vitro transcribed mRNAs of ALS and IGFBP3. IGFBP3 and ALS mRNAs were injected in mixture and their products were immunoprecipitated by antisera against ALS and IGFBP3. Contrary to the traditional reports that ALS interacts only with IGF-bound IGFBP3, this study shows that ALS is capable of forming a binary complex with IGFBP3 in the absence of IGF. When cross-linked by disuccinimidyl substrate, band representing ALS-IGFBP3 complex was evident on the PAGE. IGFBP3 movement was monitored according to the distribution between the hemispheres. Following a localized translation in the vegetal hemisphere, IGFBP3 was shown to remain in the vegetal half in the presence of ALS. Different from wild type IGFBP3, however, mutant IGFBP3 freely diffused into the animal half despite the presence of ALS. Taken together, this study suggests that ALS may play an important role in sequestering IGFBP3 polypeptides via the intermolecular aggregation. Studies using this heterologous model will lead to a better understanding of the IGFBP3 and ALS assembling into the ternary structure and circulating IGF system.