• BMB Reports
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• 제30권1호
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• pp.80-84
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• 1997

The synthesis and secretion of human angiogenin in E. coli by the natural leader sequence has been studied. We constructed a recombinant plasmid containing human angiogenin cDNA which encompassed all the coding region including leader sequence required for secretion. The recombinant plasmid was introduced into a suitable E. coli host. The angiogenin was detected in the culture medium and periplasm upon the induction of gene expression. The molecular weight of the secreted angiogenin was identical to that of authentic angiogenin purfied from human plasma when estimated by SDS-PAGE and immunoblotting. showing that the natural leader sequence was recognized and processed by the secretion machinery of E. coli. The angiogenin concentration in the culture medium reached a maximum within 2 h when expressed at $37^{\circ}C$ with 0.02~2 mM IPTG. In contrast, the expression level increased gradually over time up to 11 h at $23^{\circ}C$ with 0.002~2 mM IPTG and at $37^{\circ}C$ with 0.002 mM IPTG.

• 대한의생명과학회지
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• 제17권1호
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• pp.85-88
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• 2011

Tumor angiogenesis, which is essential for tumor growth and tumor metastasis, depends on angiogenic factors produced by tumor cells and/or infiltrating cells such as endothelial cells and immune cells in tumor tissue. Previously, we reported that licochalcone A (LicA), an important bioactive compound of Glycyrrhiza inflate, suppresses angiogenesis, tumor growth and metastasis. In this study, we evaluated the effect of LicA on angiogenin production in colon cancer cells because angiogenin is an essential factor to regulate angiogenesis and tumor progression. When we examined the angiogenin levels in three human colon cancer cells, HT-29, SW480 and Caco-2, LicA treatment significantly reduced the amounts of angiogenin among three cancer cell lines. In an in vivo study in which mice were implanted with HT-29 cells, oral administration of LicA reduced angiogenin in tumor tissues when compared with vehicle-administered mice. These results suggest that reduced angiogenin in response to LicA treatment may play essential role to inhibit tumor growth, angiogenesis as well as metastasis.

• 한국자기공명학회논문지
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• 제10권2호
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• pp.155-162
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• 2006

Angiogenin is unique among angiogenic molecules in that it is a member of the pancreatic ribonuclease superfamily and, in fact, is a ribonucleolytic enzyme. Its enzymatic activity is extremely weak compared to that of the digestive RNases but is critical for its capacity to induce neovascularization. In this study, we completed the backbone resonance assignment of bovine angiogenin using triple resonance NMR experiments of $^{15}N\;and/or\;^{13}C$ isotope labeled protein and investigated the chemical shift variation upon binding with inhibitor phosphate ion and determine the phosphate binding site.

• 대한화학회지
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• 제38권10호
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• pp.769-773
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• 1994

우유에서 추출한 안지오제닌을 사용하여 Xanthomonas celebensis 5S rRNA의 고차원 구조를 조사하였다. 안지오제닌은 5S rRNA의 단일가닥 부분에 있는 피리미딘 염기들의 먼 쪽으로 있는 3' P-O 에스테르 결합들만을 절단하였다. pH 7.0이고 10 mM의 $Mg^{2+}$이 있을 때 안지오제닌은 5S rRNA의 d 고리에서만 작용하였지만 $Mg^{2+}$이 없을 때는 e 고리를 제외한 모든 고리들(a, b, c, d 고리들)에서 작용하였다. $Mg^{2+}$이 없을 때 $U_{74}$-$G_{75}$와 $U_{77}$-$A_{78}$, $U_{103}$-$A_{104}$ 결합들이 pH 7.0과 pH 3.5에서 모두 안지오제닌의 작용에 매우 민감하였다. 한편 pH 3.5이고 $Mg^{2+}$이 없을 때 안지오제닌이 a 고리의 $C_{17}$-$G_{18}$과 b고리의 $U_{55}$-$G_{56}$ 결합들을 강하게 절단하였다. 이러한 결과들에서 우리는 다음과 같이 결론을 내릴 수 있다. 첫째, 안지오제닌을 5S rRNA의 삼차구조 분석에서의 탐침의 하나로 사용할 수 있음을 알았다. 둘째, 5S rRNA의 d고리의 구조는 $Mg^{2+}$과 $H^+$농도에 따라 변하기 쉽다는 것을 알았다.

• Journal of Animal Science and Technology
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• 제46권1호
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• pp.77-82
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• 2004

본 연구는 우유의 angiogenin(ANG) 생리활성기능을 조사하기 위한 전단계로 ANG 정제 방법을 확립하기 위하여 실시하였다. 우유로부터 ANG을 CM-Sepharose ion exchange chromatography, HPLC, Gel-filtration의 3단계 방법을 이용하여 정제하였다. 1단계인 CM-Sepharose ion exchange chromatography를 수행 한 결과 0.6 M NaCl 농도의 36번 분획에서 ANG의 밴드를 볼 수 있었고 2단계인 Mono-S 컬럼을 이용하여 HPLC를 수행한 결과에서는 1.0 M에서 하나의 peak가 분리되었고 전기영동 결과는 LF와 ANG 두 가지 성분이 함유되어 있음을 확인하였다. 마지막 단계인 Gel-filtration을 수행한 결과 ANG이 단일 밴드로 분리되었다. 우유 10 $\ell$로부터 약 80 ${\mu}\ell$의 ANG를 얻었고 정제된 ANG을 N-말단 아미노산 배열을 분석한 결과 AQDDYRYIHFLTQHY로 밝혀졌다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제32권1호
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• pp.8-18
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• 2006

Purpose: The purpose of this study was to verify that the expressions of angiogenin, transforming growth factor-beta(TGF-${\beta}$), vascular endothelial growth factor(VEGF), human apurinic/apyrimidinic endonuclease(APEX) and tumor necrosis factor-alpha(TNF-${\alpha}$) were associated with the tumorigenesis of the oral squamous cell carcinoma(OSCC). Materials and Methods: Fifty-one samples of OSCC and fifteen normal oral mucosae were obtained to analyze the expression levels of above five factors. mRNA expressions were quantified by the quantitative competitive PCR(QC-PCR) method. After 2% agarose gel electrophoresis stained with ethidium bromide, the concentration of mRNA was calculated by a digital image analysis system. The expression levels of angiogenin, TGF-${\beta}$, VEGF, APEX and TNF-${\alpha}$ were compared by unpaired Student's t-tests between cancer and normal tissues. We analyzed statistically to find the cut-off values that would be useful as diagnostic markers, and the linear regression analysis between every two factors of these five factors by SAS system. Results: All of these five factors (angiogenin: P<0.0037, TGF-${\beta}$: P<0.0001, VEGF: P<0.0102, APEX: P<0.0023, TNF-${\alpha}$: P<0.0074) were significantly correlated with OSCC. In the analysis to find the cut-off values for the diagnosis, we could not find any value that had a reasonable sensitivity and specificity. In the linear regression analysis, there were correlations between angiogenin and TNF-${\alpha}$, TGF-${\beta}$ and VEGF, TGF-${\beta}$ and APEX, TGF-${\beta}$ and TNF-${\alpha}$, VEGF and APEX, VEGF and TNF-${\alpha}$, APEX and TNF-${\alpha}$. Conclusion: Our results suggest that not only angiogenin, TGF-${\beta}$, VEGF, APEX and TNF-${\alpha}$ are significantly associated with the tumorigenesis, but also the close relationship between these factors might enhance the tumorigenesis of OSCC. We can not find clinical availability for diagnosis.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권1호
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• pp.1-5
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• 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.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1996년도 정기총회 및 학술발표회
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• pp.43-43
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• 1996

Bovine angiogenin (bAng) is a potent blood vessel inducing protein purified from cow milk. Fluorescence spectroscopy has been used to study the interaction of bAng with actin in 50 mM Tris-HCl, pH 7.5, and 1 mM CaCl$_2$ at 25$^{\circ}C$. Actin contains four tryptophans but bAng contains no tryptophans. A 50% decrease in intrinsic fluorescence accompanied formation of the bAng/actin complex. (omitted)

• KSBB Journal
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• 제17권2호
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• pp.153-157
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• 2002

Poly-Iysine이 tagging된 hEGF와 angiogenin(6L10ESA)의 융합단백질의 고체상 재접힘이 heparin-Sepharose colullln에서 수행되었을 때, untagging 단백질(E5h)의 기존의 액상 재접힘 방법과 비교하여 재접힘 수율은 약 13배 정도 증가하였다. 게다가 poly-Iysine tagging된angiogenin은 heparin에 친화도를 높여주므로 2.5배에서 3배 정도의 흡탁 수율이 증가한다. 재접힘 수율은 고체상 반응으로 인해 높은 재현성을 보였다. 재접힘 공정시간은 대략 8배 단축되었다. 고체상 재전힘된 단백질은 자신의 생물학적 역가를 유지하였다. 따라서 이 연구는 고체상 재접힘 방법이 분자간의 상호작용을 억제하여 응집현상을 현저히 줄였기 때문에 기인한 결과로 생각된다. 따라서 응집으로 인한 재접힘 수율이 낮은 단백질의 재접힘 긍정에 고체상 재접힘 공정을 사용하면 높은 재접힘 수율을 얻을 수 있다.

• BMB Reports
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• 제29권4호
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• pp.353-358
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• 1996

Bovine angiogenin (bAng) is a potent blood vessel inducing protein purified from cow In ilk. fluorescence spectroscopy has been used to study the interaction of bAng with actin in 50 mM Tris-HCl pH 7.5, and 1 mM $CaCl_2$ at $25^{\circ}C$. Actin contains four tryptophans but bAng contains no tryptophans. A 50% decrease in intrinsic fluorescence accompanied formation of the bAng/actin complex. By contrast, the interaction of RNase A, a homologous protein to bAng, with actin results in about 10% quenching of the fluorescence. Fluorescence titration experiments were performed by adding increasing concentrations of bAng (0~1.0 ${\mu}M$) to a constant concentration of actin (0.1 ${\mu}M$), and the dissociation constant $K_d$ for the bAng/actin complex and the stoichiometry n were measured as $20{\pm}1$ nM and $1.0{\pm}0.1$ respectively. These results suggest that the interaction between bAng with actin is specific and that quenching of actin fluorescence has occurred in the bAng/actin complex. The bAng binding sites of actin are discussed in the results of this study, and we propose that Trp-80 in the small domain of bovine actin is responsible for the bAng/actin binding.