Biomedical subjects

Kyung-Hwan Jung

Publications and source records attributed to Kyung-Hwan Jung.

8 recordsLinked to original sources

PubMed2006

Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation.

In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.

Angiogenesis Inhibitors↗

PubMed2005

Extended operation of a pressurized 75-L bioreactor for shLkn-1 production by Pichia pastoris using dissolved oxygen profile control.

In this study, a dissolved oxygen (DO)-stat fed-batch process was conducted in a pressurized 75-L bioreactor, resulting in the production of the short version of human leukotactin-1 (shLkn-1) using Pichia pastoris as the host, with control of the DO-stat profile and an extension of the recombinant shLkn-1 production phase. By regulation of the exhaust-gas valve, we were able to maintain the vessel pressure at up to 120 kPa, in order to overcome DO limitations associated with the use of the DO-stat. The lowest DO value was adjusted by varying the feed pump speed, allowing us to control the DO-stat profile. This principle was successfully applied to both glycerol feeding during the growth phase and methanol feeding for the induction of shLkn-1. The extension of the methanol induction phase to a total of 192 h of culture time resulted in a shLkn-1 concentration of 2.5 g/L, and a total of 102 g of cumulative production. During this extended induction period, the C-terminal residue of shLkn-1 was truncated and this was confirmed by both reverse-phase HPLC and mass spectrometry.

Bioreactors↗

PubMed2005

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V).

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.

Angiogenesis Inhibitors↗

PubMed2004

Paradoxical effects of elastase inhibitor guamerin on the tissue repair of two different wound models: sealed cutaneous and exposed tongue wounds.

Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.

Animals↗

PubMed2004

Crystallization and preliminary X-ray crystallographic study of the leech protease inhibitor guamerin and its complex with bovine pancreatic chymotrypsin.

Guamerin, a small peptide inhibitor of the serine protease from Hirudo nipponia, was expressed in yeast and crystallized using the vapor diffusion method, with MPD as precipitant. The crystal was found to belong to the monoclinic P2(1) space group with unit cell parameters a=136.06, b=206.59, c=227.39 A, beta=105.03 degrees. The guamerin/bovine pancreatic chymotrypsin complex was also crystallized using PEG 8K as precipitant. The space group was identified as P2(1)2(1)2(1) with unit cell parameters of a=44.01, b=44.30, c=122.47 A. The diffraction data of the complex were collected up to a resolution of 2.4 A using a synchrotron-radiation source under cryogenic condition.

Animals↗

PubMed2004

Production of the kringle fragments of human apolipoprotein(a) by continuous lactose induction strategy.

A novel lactose induction strategy for the production of rhLK68, the kringle fragments of human apolipoprotein(a) (apo(a)) as a novel anti-angiogenic protein, was investigated. A scale-up of the production was accompanied by a decrease in expression level, and severe aggregation occurred during the solubilization of rhLK68 from the inclusion body during a conventional single introduction of lactose. To overcome this problem, a continuous induction strategy was applied where lactose was mixed with glycerol and fed continuously in a dissolved oxygen (DO)-stat manner. With the sub-optimal feed medium consisted of 1:50 of lactose/glycerol (w/w), the expression level reached 16% of the total cellular protein, which was 1.6-fold higher than that obtained from the conventional lactose induction. Moreover, the solubilization yield of rhLK68 from the inclusion body increased from 30 +/- 5 to 85 +/- 3% compared to the conventional single introduction of lactose. This result suggests that the continuous lactose induction strategy beneficially influenced the expression level of rhLK68 and the quality of its inclusion body.

Apolipoproteins↗

PubMed2004

Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases.

Apolipoprotein(a) [apo(a)] contains the largest numbers of kringle domains identified to date. Of these, apo(a) kringle V shows significant sequence homology with plasminogen kringle 5, which is reported to be a potent angiogenesis inhibitor. To determine the effects of apo(a) kringle V on angiogenesis, it was expressed as a soluble protein (termed rhLK8) in Pichia pastoris and its in vitro and in vivo anti-angiogenic properties were examined. rhLK8 inhibited the migration of human umbilical vein endothelial cells in vitro in a dose-dependent manner. This function was associated with the down-regulation of the activation of focal adhesion kinase and the inhibition of the consequent formation of actin stress fibers/focal adhesions. rhLK8 also inhibited new capillary formation in vivo, as assessed by the chick chorioallantoic membrane assay and the Matrigel plug assay. These results indicate that rhLK8 may be an effective angiogenesis inhibitor both in vitro and in vivo.

Actins↗

PubMed2003

Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a).

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.

Allantois↗