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果胶是植物中的一种酸性多糖物质,主要存在于植物的细胞壁和细胞内层,为内部细胞的支撑物质。果胶类多糖包括三大类:同型半乳糖醛酸聚糖(HG),鼠李糖半乳糖醛酸聚糖Ⅰ(RG-Ⅰ)和鼠李糖半乳糖醛酸聚糖Ⅱ(RG-Ⅱ)[1-2]。其中RG-Ⅱ至少有12种不同成分的糖基残基组成,而2-酮-3-脱氧辛糖酸(KDO)是RG-Ⅱ成分中很少见的八碳糖[3],其在进化过程中也很保守,对花粉管的生长和伸长具有一定的作用[4-5]。KDO的合成部位为细胞溶胶质,合成过程中涉及到5种酶[6]。脱氧辛糖酸-8-磷酸合酶(3-Deoxy-D-manno-octulosonate 8-phosphate synthase,KDO8PS,EC)是参与KDO合成代谢的关键酶之一,催化浓缩磷酸烯醇式丙酮酸与α-阿拉伯糖5磷酸产生2-酮-3-脱氧辛糖8磷酸(3-Deoxy-D-manno-octulosonate8-phosphate),并释放无机磷酸(Pi)。产物2-酮-3-脱氧辛糖-8磷酸是2-酮-3-脱氧辛糖酸的前体。最初研究发现,在所有的革兰氏阴性菌中(Gˉ),2-酮-3-脱氧辛糖酸是脂多糖组分中必需的八碳糖,它连接类脂A与O-特异侧链共同嵌入细胞壁的外膜上,其中类脂A是内毒素的物质基础,O-抗原的多样性决定了Gˉ表面抗原决定簇的多样性,脂多糖(LPS)具有控制着某些物质进出细胞的部分选择性屏障功能[7]。此后又发现,2-酮-3-脱氧辛糖酸也是高等植物细胞壁以及一些绿色藻类植物细胞壁多糖的成分[3, 8]。大肠埃希菌Escherichia coli中,KDO8PS晶体结构已经被解析,它是一个不对称同源四聚体,其中每个单体都含有(β/α)8桶状折叠[9],在拟南芥Arabidopsis thaliana中分离到2个编码KDO8PS的旁系同源基因AtkdsA1和AtkdsA2[10],相对于茎干、幼嫩的叶、成熟的叶,成熟花中AtkdsA2基因的表达量明显增高,对花粉管的生长和伸长具有一定的作用,并发现KDO8PS在水溶液中主要是以二聚体形式存在[11]。但是由于在植物中KDO8PS晶体结构尚未解析,酶功能及催化作用机制、不同生物来源KDO8PS酶的催化特性等科学问题仍需深入研究。中国是竹子大国,竹产业的发展蕴藏着巨大的经济效益,其中食用竹笋拥有丰富的营养价值,被公认为是最佳的绿色食品、经济价值高[12]。在众多竹类中,毛竹Phyllostachys edulis是中国竹类植物中分布范围最广、栽培面积最大、蓄积量最多、经济价值最高的一个材用和笋用竹种,在中国林业生产中占有非常重要的地位。本研究拟克隆毛竹脱氧辛糖酸-8-磷酸合酶基因,分析其在毛竹不同组织表达特异性,进行进化系统分析和在原核细胞中过量表达,经过亲和层析和分子筛层析纯化得到高纯度蛋白,用于培养晶体。实验结果显示:该蛋白在溶液中是以二聚体形式存在,并筛选出微晶,为解析其晶体结构和揭示其酶催化机制奠定基础。
Expression, purification, and crystallization of KDO8PS from Phyllostachys edulis
doi: 10.11833/j.issn.2095-0756.2014.04.004
- Received Date: 2013-10-21
- Rev Recd Date: 2014-01-13
- Publish Date: 2014-08-20
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Key words:
- forest tree breeding /
- Phyllostachys edulis /
- 3-deoxy-D-manno-octulosonate (KD0)8-phosphate synthase (KDO8PS) /
- tissue-specific expression /
- prokaryotic expression /
- protein crystallization /
- crystal
Abstract: 3-deoxy-D-manno-octulosonate (KDO) 8-phosphate synthase (KDO8PS)[EC 4.1.2.16] is the rate-limiting enzyme in the KDO biosynthetic pathway. In this study, the KDO8PS gene (3-deoxy-d-manno-octulosonate-8-phosphate synthase) was cloned from fresh Phyllostachys edulis seedlings using Reverse transcription polymerase chain reaction (RT-PCR). Amino acid sequence alignment analysis of the KDO-8P synthase from different organisms and tissue specific expression analysis by semi-quantitative RT-PCR were conducted. After, PeKDO8PS was recombined into the expressed pET-28a vector and over expressed in Escherichia coli. Then the fusion protein was purified through using a two-steps purification strategy including nickel affinity and size exclusion chromatography (SEC). Results showed that the open reading frame was 876 bp encoding a protein consisting of 291 amino acid residues. The PeKDO-8P synthase had high similarity with KDO-8P synthase from plants such as AtkdsA2 from Arabidopsis thaliana, while low identities from microorganism. The tissue specific expression analysis showed much higher gene expression in roots than in stems and leaves which was similar to AtkdsA2 from Arabidopsis thaliana. Finally, the chromatography analysis showed that the PeKDO8PS protein mainly existed as dimmer in 30 mmmol·L-1 Tris-HCl pH 8.0, 200 mmol·L-1 NaCl, from which the growth of preliminary screening protein crystallization condition was obtained. These works provide the first step for its structure determination.
Citation: | ZHANG Fengxue, XU Yingwu, ZHANG Zhijun, XIAO Dongchang, WANG Chaoli, QU Yaping. Expression, purification, and crystallization of KDO8PS from Phyllostachys edulis[J]. Journal of Zhejiang A&F University, 2014, 31(4): 515-520. doi: 10.11833/j.issn.2095-0756.2014.04.004 |