Isolation of a large thaumatin-like antifungal protein from seeds of the Kweilin chestnut Castanopsis chinensis
Isolation of a large thaumatin-like antifungal protein from seeds of the Kweilin chestnut Castanopsis chinensis
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DOI：
10.1016/s0006-291x(02)02998-4
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发表时间：
2003-02-07
影响因子：
3.1
通讯作者：
Ng, TB
中科院分区：
文献类型：
--
作者：
Chu, KT;Ng, TB
从板栗（Castanopsis chinensis Hance）的种子中纯化了一种具有N-末端序列的蛋白质，该蛋白质与菜豆和猕猴桃的奇异果类奇异果甜蛋白（thaumatin-like protein，TLPs）相比，与其他TLPs的同源性要小得多，并且具有30 kDa的分子量，这比先前报道的TLPs的分子量高得多。蛋白质未吸附在10 mM Tris-HCl缓冲液（pH 7.3）中的DEAE-纤维素上，并吸附在相同缓冲液中的Affi-凝胶蓝色凝胶上，吸附在10 mM乙酸铵缓冲液（pH 4.5）中的CM-纤维素上，以及吸附在20 mM乙酸铵缓冲液（pH 5.5）中的Mono S上。在前三种层析介质上分级分离后获得高度纯化的蛋白质制剂。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中出现单一条带（30 kDa），在Superdex 75凝胶过滤中通过快速蛋白质液相色谱出现单一峰（30 kDa）。TLP对灰葡萄孢、尖孢镰刀菌、花生球腔菌和梨轮孢菌具有抗真菌活性，对尖孢镰刀菌的IC 50为0.5 μ M。结果表明，苦槠类TLPs的抗真菌活性高于菜豆类和猕猴桃类TLPs。oxysporum和M.花生。经40 ℃处理10 min后，其抑菌活性基本不变，60 ℃处理后抑菌活性降低，80 ℃处理后抑菌活性消失。用胰蛋白酶（酶：底物比1：100）在37 ℃处理1h后，其抗真菌活性有所下降，但仍有一定的活性。锥属TLP对HIV-1逆转录酶的抑制活性（IC_（50）= 1.6 μ M）比猕猴桃TLP（IC_（50）大于或等于27 μ M）强得多。每kg板栗种子可获得20 mg的锥属TLP。(C)2003 Elsevier Science（美国）。All rights reserved.
A protein with an N-terminal sequence showing a much lesser extent of homology than French bean and kiwi fruit thaumatin-like proteins (TLPs) to other TLPs, and possessing a molecular mass of 30 kDa which is considerably higher than those of previously reported TLPs, has been purified from the seeds of the chestnut Castanopsis chinensis Hance. The protein was unadsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.3), and adsorbed on Affi-gel blue gel in the same buffer, on CM-cellulose in 10 mM ammonium acetate buffer (pH 4.5), and on Mono S in 20 mM ammonium acetate buffer (pH 5.5). A highly purified protein preparation was obtained after fractionation on the first three chromatographic media. Castanopsis TLP appeared as a single band (30 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a single peak (30 kDa) in gel filtration on Superdex 75 by fast protein liquid chromatography. The TLP exerted antifungal activity against Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola, and Physalospora piricola, with an IC50 of 0.5 muM against E oxysporum. Castanopsis TLP was more potent than French bean and kiwi fruit TLPs in its antifungal activity toward F. oxysporum and M. arachidicola. The antifungal activity of Castanopsis TLP remained essentially unaltered after incubation at 40degreesC for 10min, was reduced after incubation at 60degreesC, and disappeared after treatment at 80degreesC. The antifungal activity underwent a decline after treatment with trypsin (enzyme: substrate ratio 1:100) at 37degreesC for 1 h but some activity remained. Castanopsis TLP exhibited a much more potent inhibitory activity on HIV-1 reverse transcriptase IC50 = 1.6 muM) than kiwi fruit TLP (IC50 greater than or equal to 27 muM). Castanopsis TLP was obtained with a yield of 20 mg from 1 kg chestnut seeds. (C) 2003 Elsevier Science (USA). All rights reserved.