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RISS 인기검색어
- 검색키워드
- 저자 : Byun, Tae-ho (검색결과 391 건)
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Byun, Jong-Seon Kangwon National University 2008 국내석사
Vasoactive Intestinal Peptide (VIP), a 28-amino acid peptide, is a neuropeptide with a potent anti-inflammatory and neuroprotective effect. The pharmacology of VIP has spectacularly grown, and VIP itself, as well as more stable VIP-derived agents, have been used or proposed as efficient therapeutical treatments of several disorders, specifically inflammatory and autoimmune diseases, such as septic shock, rheumatoid arthritis, multiple sclerosis, Crohn's disease, and autoimmune diabetes. Previous studies have indicated the requirement of astrocyte for VIP-relative neuroprotection and involved proteins such as activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP), glial proteins mediating the VIP neuroprotective effects. In this study, it is aimed to clone the human-VIP and investigate whether it mediates the neuroprotective activity against the glucocorticoid-induced cellular stress. Therefore, I cloned the VIP using the genetic manipulation from human thalamus cDNA, then cloned human-VIP gene into the pET32a(+) expression vector and transformed into the Rosetta (DE3) strains. The recombinant protein expression was confirmed by SDS-PAGE and immunoblotting. In SDS-PAGE, a thick protein band was visualized after the gel was stained with Coomasie brilliant blue. The expressed recombinant proteins were purified by affinity chromatography and gel filtration chromatography. And I obtained recombinant protein as an inclusion body form. So, I removed the inclusion body using protease such as enterokinase. The remaining impurities were removed with ultra filtration membrane. VIP was treated in dexamethasone pretreated primary cultured astrocyte. Then alteration of gene expression levels were analysed by micro-array. And VIP receptor activity was measured by cAMP assay. As a result, neurotrophic factors such as GDNF and BDNF were increased and apoptotic mediators such as caspase 3 and caspase 9 were decreased by VIP peptide in dexamethasone pretreated primary cultured astrocyte. And I did not obtained result that I expected from cAMP assay. I’ll try to test more. This result is not perfect to understand the VIP mediating neuroprotective effect against dexamethasone?induced cellular stress. But, I can found that neurotrophic factor, CREB was increased and apoptotic mediators were decreased by VIP. Therefore, I need to further study about VIP’s signaling mechanisms. 28개의 아미노산으로 구성된 펩타이드인 Vasoactive Intestinal Peptide(VIP)는 항 염증과 신경세포 보호효과를 가진 neuropeptide이다. VIP와 VIP 유도체는 염증반응 이나 전염성 자극, 류마티스염, 광역적인 병변 및 Crohn’s 질병과 같은 자가면역 질병의 효과적인 치료제로서 처방되거나 사용 되어 왔다. 최근의 연구에서는 ADNF 와 ADNP 같은 glial 단백질들이 VIP에 의한 신경세포 보호작용을 매개한다고 보고되고 있다. 그리하여 본 연구는 인간 VIP를 cDNA library로부터 동정하고 그것이 당질스테로이드에 의해 발생되는 세포의 스트레스에 대하여 신경보호효과를 매개하는 것을 확인하고자 하였다. 그리하여 인간 시상하부로부터 유래된 cDNA로부터 유전자 조작법을 통하여 인간 VIP 유전자를 동정하였고 이를 단백질 발현 벡터인 pET32a(+)에 도입하여 박테리아에 형질전환 시켜 재조합 단백질을 만들고자 하였다. 인간 VIP 유전자를 박테리아에 과 발현 시킨 후 SDS-PAGE와 Immunoblotting을 통해 재조합 단백질의 발현을 확인하고자 하였다. SDS-PAGE 상에서 쿠마시 블루 염색법을 통해 재조합 단백질 발현을 밴드로서 확인하였다. 재조합 단백질을 정제하기 위해 친화 크로마토그래피 와 젤 거르기 크로마토그래피를 시행하였다. 이 방법을 통해서 inclusion body를 가지고 있는 재조합 인간 VIP를 얻었다. 그래서 이 inclusion body를 제거하기 위하여 enterokinase라는 단백질 절단효소를 처리 하였고, 또한 VIP 외의 다른 단백질들을 제거하기 위해 ultra-filtration membrane을 사용하였다. 이렇게 해서 얻은 VIP 단백질은 dexamethasone을 전처리 한 일차배양 성상세포에 처리하였다. 그런 후에 micro-array 법을 통해 유전자의 발현의 변화를 분석하였고 cAMP assay를 통해 receptor의 활성을 측정하였다. 이에 따른 결과는 VIP에 의해 neurotophic 인자인 GDNF와 BDNF의 발현이 증가 하였고, caspase 3 와 caspase 9 같은 apoptosis 인자들은 감소 하는 것을 확인 하였다. 그리고 receptor 활성을 측정하기 위해 cAMP assay를 한 결과는 예상했던 것 과 다른 결과를 얻게 되었다. 좀 더 실험을 진행해야 할 것이다. 이 논문의 결과로는 dexamethasone에 의해 일어나는 세포 스트레스에 대해 VIP가 보호효과를 매개한다고 정확하게 이해할 수는 없다. 그러나 나는 VIP에 의해서 neurotrophic factor 와 transcription factor인 CREB의 발현 증가, 그리고 apoptosis 인자들이 감소하는 것을 확인함으로써 가능성을 얻었으며, 앞으로 VIP의 메커니즘에 대해 좀더 많은 연구가 진행되어야 할 것이다.
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Byun, Sungmin Kwangwoon University Graduate School 2015 국내석사
Lysine cyclodeaminase (LCD; EC: 4.3.1.28) is a β-nicotinamide adenine dinucleotide-dependent enzyme that catalyzes the β-deamination of L-lysine to produce L-pipecolate. L-pipecolate, also known as L-homoproline, is an immunosuppressant and can be incorporated into multiple secondary metabolite products. Recombinant lysine cyclodeaminase from Streptomyces pristinaespiralis (spLCD) has been successfully expressed in E. coli. Among various substrates with different carbon lengths and enantiomeric statuses, L-lysine was found to be the best substrate for spLCD. We also examined the reaction conditions (buffer type, pH and temperature) to yield a high concentration of L-pipecolic acid. Although spLCD was found highly enantioselective toward L-lysine, its enzymatic activity as well as thermostability was seriously decreased under acidic pH conditions and at temperatures higher than 60°C, respectively. A final conversion of L-lysine to L-pipecolate of over 90% was achieved under optimal reaction conditions of 200 mM PIPES buffer, pH 7.0, and a temperature of 60°C. Despite the many advantages of enzymatic reactions, they are often limited by their own catalytic repertoire. By increasing the number of structures and the mechanism information, rational enzyme engineering of substrate specificity has broadened enzymatic usage. Pipecolic acid is an unnatural amino acid mostly used for pharmaceutical purposes. Pipecolic acid has two types of enantiomers with different roles in the synthesis of drugs. The development of efficient catalytic methods for the production of enantiopure pipecolic acid is currently a crucial topic of research. Few chemo- or biosynthetic methods have been proposed for the synthesis of pure enantiomers; however, enzymatic conversion of the chirality of pipecolic acid has not been demonstrated because no pipecolic acid racemase has been reported yet. In this work, we attempted to engineer pipecolic acid racemase activity into Trypanosoma cruzi proline racemase (TcPRAC) for the enzymatic synthesis of D-pipecolic acid from L-pipecolic acid. For the binding of pipecolic acid (C6 ring) into the active site of TcPRAC, which was optimized for the original substrate proline (C5 ring), four bulky aromatic residues (Phe102, Phe120, Phe220, and Phe 290) of TcPRAC were mutated to smaller hydrophobic residues. Among the mutants, six single-point mutants (F102A, F102I, F102L, F102V, F290L, and F290V) exhibited significant racemase activity against L-pipecolic acid. The most efficient variant, F102V, showed 74% racemization. Molecular docking simulations revealed that lowering the binding energy of L-pipecolic acid to the active site was important for achieving high racemization activity of TcPRAC mutant proteins.
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