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水稻cDNA c73片段在大肠杆菌中的表达及其产物的纯化

唐威华, 仇子龙, 王宗阳, 张景六, 洪孟民

中科院上海植物生理研究所.上海 200032

摘要

曾以水稻蜡质基因5^调控区内一段31bp片段为探针,用酵母单杂交法从水稻cDNA文库中筛选出若干个其编码的蛋白可能与此31bp片段结合的cDNA克隆,现将其中的pC73克隆中的插入片段c73连接到含His_(6)的表达载体pET28-c(+)上,在大肠杆菌BL21(DE3)中进行诱导表达,并用Ni-NTA树脂纯化得到预期的融合表达产物。在合适的诱导表达条件下,融合表达产物主要以可溶形式存在于大肠杆菌细胞内;表达量占到大肠杆菌总蛋白的10%左右,经Ni-NTA树脂亲和层析纯化得到的产物纯度达95%,可供进一步研究之用。

关键词： 水稻; 转录因子; DNA结合蛋白 His_(6)融合表达; Ni-NTA树脂亲和层析

Expression of a rice cDNA c73 in E. coli cells and purification of its fusion protein

Tang Weihua, Qiu Zilong, Wang Zongyang, Zhang Jingliu, Hong Mengmin

Shanghai Institute of Plant Physiology,Chinese Academy of Sciences.Shanghai 200032

Abstract

A rice cDNA fragment c73, one of the candidates for the sequence encoding a DNA-binding protein, has been isolated from a rice cDNA library, using a 31 bp DNA fragment in the 5^-upstream region of rice waxy gene as a probe. To isolate and purify the gene product, the c73 DNA was cloned into a His_(6)-fusion expression vector pET28-c(+). After being transformed into E. coli BL21(DE3), the expected fusion protein encoded by c73 fragment was expressed by induction by IPTG, and purified using Ni-NTA affinity chromatography.

Key words: Rice;Transcription factor;DNA-binding protein;His_(6) fusion expression

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引用本文：

唐威华, 仇子龙, 王宗阳, 张景六, 洪孟民. 水稻cDNA c73片段在大肠杆菌中的表达及其产物的纯化[J]. 生理学报 1999; 51 (4): .

Tang Weihua, Qiu Zilong, Wang Zongyang, Zhang Jingliu, Hong Mengmin. Expression of a rice cDNA c73 in E. coli cells and purification of its fusion protein. Acta Physiol Sin 1999; 51 (4): (in Chinese with English abstract).