• Title/Summary/Keyword: Angiostatin-binding protein

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Identification and Characterization of a Novel Angiostatin-binding Protein by the Display Cloning Method

  • Kang, Ha-Tan;Bang, Won-Ki;Yu, Yeon-Gyu
    • BMB Reports
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    • v.37 no.2
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    • pp.159-166
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    • 2004
  • Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of $3.4{\times}10^{-7}\;M$. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.

Soluble Expression of Human Angiostatin and Endostatin by Maltose Binding Protein (MBP) Fusion in E. coli (Maltose Binding Protein 융합단백질에 의한 인간유래의 앤지오스타틴과 앤도스타틴의 대장균에서 수용성 단백질발현)

  • Paek, Seon-Yeol;Choi, Shin-Geon
    • Journal of Industrial Technology
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    • v.28 no.B
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    • pp.59-63
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    • 2008
  • Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

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In vitro Interaction of Recombinantly Expressed Kringle 5 (rK5) with Ras Guanine Nucleotide Dissociation Stimulator-like Factor (Rgl2)

  • Lee, Jung-Whoi;Kim, Sun-Hee;Park, Yong-Sung;Woo, Je-Wan;Lim, Dong-Yeol;Lee, Kyung-Hee
    • Bulletin of the Korean Chemical Society
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    • v.25 no.12
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    • pp.1863-1868
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    • 2004
  • Kringle 5 (K5), located outside of angiostain (K1-4) in human plasminogen, displays more potent antiangiogenic activity on endothelial cell proliferation than angiostatin itself. Using a yeast two-hybrid system in vivo, we have recently identified Rgl2 (guanine nucleotide dissociation stimulator (RalGDS)-like factor 2) as a binding protein of human K5. In order to confirm in vitro protein interaction between K5 and Rgl2, we developed bacterial recombinant expression systems for them. K5 and Rgl2 proteins were expressed in high yields and purified into pure forms with His tags and GST fusion, respectively. GST-pull down experiments clearly demonstrated that K5 interacts specifically with Rgl2 in vitro. These results indicate that Rgl2 functions as a receptor protein for K5 in vitro as well as in vivo, leading to anti-angiogenesis through regulating Ras signaling pathways.

Solid-Phase Refolding of Inclusion Body Protein in Packed Bed Adsorption and Expanded Bed Adsorption Chromatography (Packed Bed Adsorption과 Expanded Bed Adsorption 크로마토그래피를 이용한 내포체 단백질의 고체상 재접힘)

  • 최원찬;김민영;서창우;이은규
    • KSBB Journal
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    • v.18 no.6
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    • pp.500-505
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    • 2003
  • ‘LK (lipoprotein kringle) 68’is a polypeptide of a modified ansiostatin consisting of three kringle structures that might be clinically useful as a potential cancer therapeutics. It can be produced by overexpressing it as inclusion body in recombinant E. coli. In this study, solid-phase refolding processes using packed bed adsorption (PBA) and expanded bed adsorption (EBA) column were carried out to compare their refolding yields with that of the conventional, solution-phase refolding process, For the solution-phase and the PBA-mediated processes employing Q-Sepharose, washed inclusion body was used as the starting material, whereas both washed inclusion body and E. coli homogenate were used for the EBA-mediated process employing streamline DEAE. On the final recovery LK68 per unit mass of wet cell basis, the EBA- and PBA-mediated processes showed about 2.7- and 1.5-fold higher yields, respectively, than the solution-phase refolding method. The solid-phase refolded LK68 demonstrated the same Iysine binding bioactivity and the retention time in the RP-and SEC-HPLC as those of the native protein.