• Biotechnology and Bioprocess Engineering:BBE
• /
• v.7 no.1
• /
• pp.1-5
• /
• 2002

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solid-phase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below $ 30\%$, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about $88\%$. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. $4.8\%$ in the conventional refolding of the untagged fusion protein to $63.6\%$. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.

• Genomics & Informatics
• /
• v.21 no.1
• /
• pp.9.1-9.13
• /
• 2023

Matrix metalloproteinase-9 (MMP-9) is a zinc and calcium-dependent proteolytic enzyme involved in extracellular matrix degradation. Overexpression of MMP-9 has been confirmed in several disorders, including cancers, Alzheimer's disease, autoimmune diseases, cardiovascular diseases, and dental caries. Therefore, MMP-9 inhibition is recommended as a therapeutic strategy for combating various diseases. Cinnamic acid derivatives have shown therapeutic effects in different cancers, Alzheimer's disease, cardiovascular diseases, and dental caries. A computational drug discovery approach was performed to evaluate the binding affinity of selected cinnamic acid derivatives to the MMP-9 active site. The stability of docked poses for top-ranked compounds was also examined. Twelve herbal cinnamic acid derivatives were tested for possible MMP-9 inhibition using the AutoDock 4.0 tool. The stability of the docked poses for the most potent MMP-9 inhibitors was assessed by molecular dynamics (MD) in 10 nanosecond simulations. Interactions between the best MMP-9 inhibitors in this study and residues incorporated in the MMP-9 active site were studied before and after MD simulations. Cynarin, chlorogenic acid, and rosmarinic acid revealed a considerable binding affinity to the MMP-9 catalytic domain (ΔG_binding < -10 kcal/ mol). The inhibition constant value for cynarin and chlorogenic acid were calculated at the picomolar scale and assigned as the most potent MMP-9 inhibitor from the cinnamic acid derivatives. The root-mean-square deviations for cynarin and chlorogenic acid were below 2 Å in the 10 ns simulation. Cynarin, chlorogenic acid, and rosmarinic acid might be considered drug candidates for MMP-9 inhibition.

• Proceedings of the PSK Conference
• /
• 2003.04a
• /
• pp.290.3-291
• /
• 2003

To increase detection sensitivity for multi-DDT residues (o,p-/p,p-DDT, o,p-/p,p-DDE, o,o-/o,p-DDD) analysis, a highly selective sample clean-up method was introduced prior to GC/MS analysis using immunoaffinity column. The immunoaffinity matrix was prepared by coupling IgG fraction of DDT antiserum to cyanogens bromide activated Sepharose 4B. Three DDT antisera (DDA-1, DDHP-2, DDCP-3) were test for affinity column ligand that obtained by imunizing respective DDT immunogen to rabbits, and IgG was purified using protein A affinity purification. (omitted)

• KSBB Journal
• /
• v.21 no.4
• /
• pp.279-285
• /
• 2006

We investigated the effects of immobilization chemistry on the yield of immobilization and the bioactivity of the immobilized enzymes. Trypsin as a model protein and macroporous polymer beads(Toyopearl AF 650M, Tosho Co., Japan) was used as a model matrix. Four methods were used to immobilize trypsin; covalent conjugation by reductive amination(at pH 10.0 and pH 4.0) and affinity interaction via streptavidin-biotin, and double-affinity interaction via biotin-streptavidin-biotin system. The covalent conjugation immobilized $3{\sim}4$ mg/ml-gel, ca. 3-fold higher than the affinity method. However, the specific activity of the covalently(pH 10.0) and affinity-immobilized trypsin(via streptavidin-biotin) are ca. 37% and 50%, respectively, of that of the soluble enzyme(on the low-molecular-weight BAPNA substrate). When the molecular size of a substrate increased, the affinity-immobilized trypsin showed higher clavage activity on insulin and BSA. This result seemed to indicate the streptavidin-biotin system allowed more steric flexibility of the immobilized trypsin in its interaction with a substrate molecule. To confirm this, we studied the molecular flexibility of immobilized trypsin using quartz crystal microbalance-dissipation. Self-assembled monolayers were formed on the Q-sensor surface by aminoalkanethiols, and gultaraldehyde was attached to the SAMs. Trypsin was immobilized in two ways: reductive amination(at pH 10.0) and the streptavidin-biotin system. The dissipation shift of the affinity-immobilized trypsin was $0.8{\times}10^{-6}$, whereas that of the covalently attached enzyme was almost zero. This result confirmed that the streptavidin-biotin system allowed higher molecular flexibility. These results suggested that the bioactivity of the immobilized enzyme be strongly dependent on its molecular flexibility.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.2
• /
• pp.196-203
• /
• 2002

A novel process for urokinase purification was studied using p-aminobenzamidine as the ligand and sepharose 4B as the matrix. The adsorption, washing, and elution conditions were optimized by an unusual method. An adsorption buffer containing 2.5 M NaCl and $1\%$ Tween 80 facilitated the adsorption of urokinase on the affinity media and prevented contaminants from binding to the p-aminobenzamidine affinity gel. It was found that $5\%$ Tween 80 removed most of the contaminants from the affinity column. A 0.2 M glycine elution buffer containing 0.5 M NaCl (pH 3.0) was found to have a strong elution ability with a high recovery and purity of urokinase. A crude urokinase material of231 IU/mg protein from human urine was purified to 124,300 IU/mg protein with a purification factor of 538 and yield of $86.7\%$. As a result, a high purity urokinase was obtained with only a single affinity chromatography step. The purification process was successfully scaled-up to a 2-1 chromatography column. The resulting urokinase eluate could be directly lyophilized, thereby complying with Chinese pharmacopoeia (1995 version) standards.

• The Korean Journal of Malacology
• /
• v.3 no.1
• /
• pp.3-14
• /
• 1987

The electrophoretic and innunological cnalyses of organic matrices in the shells of freshwater bivalves were made in order to dlucidate the biochemical characteristics and species-specific differinces of the applied shells, The water-soluble and insoluble matrices of four species of freshwater bivalves, Andodonta fukudai, Unio douglasiae, Lanceolaria and Chrbicula fluminea, were used as analytical materials, There was non-identity in immuno affinity between anti soluble matrix(anti-Sm) and anti insoluble matrix(anti-ISM)sera against the organic matrix of Andodnta fukudai. The SMs of four species (S. fukudai, U.douglasiae, L. acrorhyncha, and C. fluminea) showed the differences in the precipitate arcs at the level of family, though ISMs did mot show differences. In the electrophoretic analysis, all foru species had two SDS-electrophoretic bands of SM, of which molecular wights appeared to be lower than 55,000, shereas the native organic matrices of foru speceis had higher molecular weighrs than those from SDS-dldctrophoresis. Only calcium ion among many ions in extrapallial fluid(EPF) caused SM to change into insoluble molecules, thus the EPF pretreated with Ca++did not form the precipitate arc when did the immuno diffusion whth anti SM serum. ISM precursor may be polymerized into macromolecules like periostracin, a precusor of periostracum, judging from the similat polymerization patterns in 0.1M Tris formate buffer(pH 3); they may be made insoluble macromolecules due to their strong natrue of hydrophobicity.

• Journal of the Korean Society for Aeronautical & Space Sciences
• /
• v.38 no.5
• /
• pp.467-476
• /
• 2010

For the very light jet aircraft design, the design baseline configuration has been selected using the logical decision making process, and the design optimization problem is formulated by considering the airworthiness regulations as design constraints. Airworthiness regulations are the minimum requirements for the safe aircraft flight and must be considered from the conceptual design stage. After carefully selecting the airworthiness constraints and the user specified requirements, a series of design making models including the affinity diagram, nested column diagram, quality function deployment (QFD), Pugh concept selection matrix, are used to find and evaluate alternative configuration baselines. From the feasible design space searching process, the best altenative design, which satisfies the airworthiness constraints while excluding the user subjective decisions as much as possible, has been successfully derived.

• Korean Journal of Environmental Agriculture
• /
• v.25 no.4
• /
• pp.371-381
• /
• 2006

New proteomic techniques have been pioneered extensively in recent years, enabling the high-throughput and systematic analyses of cellular proteins in combination with bioinformatic tools. Furthermore, the development of such novel proteomic techniques facilitates the elucidation of the functions of proteins under stress or disease conditions, resulting in the discovery of biomarkers for responses to environmental stimuli. The ultimate objective of proteomics is targeted toward the entire proteome of life, subcellular localization biochemical activities, and the regulation thereof. Comprehensive analysis strategies of proteomics can be classified into three categories: (i) protein separation via 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification via either Edman sequencing or mass spectrometry (MS), and (iii) proteome quantitation. Currently, MS-based proteomics techniques have shifted from qualitative proteome analysis via 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, toward quantitative proteome analysis. In vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes. protein-labeling tagging with isotope-coded affinity tags, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope-labeled amino acids can be in vivo labeled into live culture cells via metabolic incorporation. MS-based proteomics techniques extend to the detection of the phosphopeptide mapping of biologically crucial proteins, which ale associated with post-translational modification. These complementary proteomic techniques contribute to our current understanding of the manner in which life responds to differing environment.

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.197-203
• /
• 2001

A fusion protein, consisting of human epidermal growth factor as a recognition domain and human angiogenin as a toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably due to the opposite surface charge due to vastly different pI values of each domain. Solid-phase refolding process exploiting ionic interactions between the solid matrix and the protein was tried, but the ionic binding yield was very low regardless of the resins and pH conditions used. To provide higher affinity toward the solid matrix, six lysine residues were tagged to the N -terminus of the hEGF domain When the cation exchange resins such as heparin- or CM-Sepharose were used as the matrix, the adsorption capacity increased 2.5-3 times and the subsequent refolding yield increased nearly IS times compared to the conventional process.

• ETRI Journal
• /
• v.35 no.2
• /
• pp.311-320
• /
• 2013

In most spectral clustering approaches, the Gaussian kernel-based similarity measure is used to construct the affinity matrix. However, such a similarity measure does not work well on a dataset with a nonlinear and elongated structure. In this paper, we present a new similarity measure to deal with the nonlinearity issue. The maximum flow between data points is computed as the new similarity, which can satisfy the requirement for similarity in the clustering method. Additionally, the new similarity carries the global and local relations between data. We apply it to spectral clustering and compare the proposed similarity measure with other state-of-the-art methods on both synthetic and real-world data. The experiment results show the superiority of the new similarity: 1) The max-flow-based similarity measure can significantly improve the performance of spectral clustering; 2) It is robust and not sensitive to the parameters.