The expression of tomato prosystemin in Escherichia coli: A structural challenge.
The expression of tomato prosystemin in Escherichia coli: A structural challenge.
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DOI：
10.1006/prep.1999.1113
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发表时间：
1999-10
影响因子：
1.6
通讯作者：
J. Delano;J. Dombrowski;C. Ryan
中科院分区：
文献类型：
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作者：
J. Delano;J. Dombrowski;C. Ryan
前系统素是18个氨基酸的多肽系统素的200个氨基酸的激素原，系统素是一种系统性移动的信号，激活茄科植物中防御基因的合成以响应草食动物的攻击。番茄前系统素cDNA和蛋白质的不寻常的一级结构特征为设计表达系统以获得全长蛋白质提供了非凡的挑战。前系统素表达抑制了所用的真核和几种原核宿主的生长。Prosystemin最初是用T7 RNA聚合酶表达系统在大肠杆菌中合成的一个长度为185个氨基酸的截短蛋白。coli BL21[DE3]。发现截短是由于两个因素：（1）前系统素序列的5'编码区与表达载体的核糖体结合位点的分子内缔合和（2）刚好在位置15处的氨基酸甲硫氨酸之前存在翻译起始位点。将允许全长前系统素蛋白合成的突变引入前系统素cDNA的氨基末端5'编码区。一个199个氨基酸的重组prosystemin缺乏的N-末端甲硫氨酸从裂解物中纯化，并通过N-末端氨基酸序列和免疫印迹分析确认。
Prosystemin is the 200-amino-acid prohormone of the 18-amino-acid polypeptide called systemin, a systemic mobile signal that activates the synthesis of defense genes in solanaceous plants in response to herbivore attacks. The unusual primary structural features of the tomato prosystemin cDNA and protein provided an extraordinary challenge in devising an expression system to obtain the full-length protein. Prosystemin expression inhibited the growth of a eukaryotic and several prokaryotic hosts used. Prosystemin was initially synthesized as a truncated protein of 185 amino acids in length using a T7 RNA polymerase expression system in E. coli strain BL21[DE3]. The truncation was found to be due to two factors: (1) the intramolecular associations of the 5' coding region of the prosystemin sequence with the expression vector's ribosome binding site and (2) the presence of a translation start site just prior to the amino acid methionine at position 15. Mutations that permitted the synthesis of the full-length prosystemin protein were introduced into the amino-terminal 5' coding region of the prosystemin cDNA. A 199-amino-acid recombinant prosystemin lacking the N-terminal methionine was purified from lysates and confirmed by N-terminal amino acid sequence and immunoblot analysis.