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SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因

唐威华, 张景六, 王宗阳, 洪孟民

中科院上海植物生理研究所.上海 200032

摘要

研究一个His-tag融合蛋白P73-His时,首先用SDS-PAGE法测得其分子量确实比理论计算值大,然后对其进行C末端氨基酸顺序测定、电喷雾质谱分析,结果证实其实际分子量与理论值一致。酶切去除包括His-tag在内的部分肽段使SDS-PAGE法测量蛋白分子量的偏差大大降低,证实His-tag确实是造成偏差的原因之一。推测由于His-tag中的碱性氨基酸的作用造成蛋白在SDS-PAGE中迁移变慢,而导致偏差。这一现象值得引起有关研究者的注意。

关键词： His-tag/Ni-NTA 融合蛋白; SDS-PAGE; 电喷雾质谱; 分子量

The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE

Tang Weihua, Zhang Jingliu, Wang Zongyang, Hong Mengmin

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

Abstract

When applying SDS-PAGE method to determine the size of a protein fused with His-tag at the N terminus (P73-His), we also found that the molecular weight of this protein was much higher than that of the value calculated from its amino acid sequence deduced from the encoding cDNA. The results of C-terminal amino acid sequencing and electrospray mass spectrometry analysis of this protein confirmed that its molecular weight was consistent with the calculated data. After a 17 amino acid peptide including the His-tag in N-terminal was excised from the fusion protein by digestion with the proteolytic enzyme thrombin, the deviation of molecular weight of this protein determined by SDS-PAGE was reduced. These results indicated that the application of SDS-PAGE as the determination of the molecular weight of a fusion protein with His-tag really caused deviation, which was related to the basic amino acid residues of His-tag which might retard the mobility of the fusion protein bands in SDS-PAGE. This phenomenon is worthy of notice to researches using His-tag fusion protein.

Key words: His tag/Ni NTA fusion protein;SDS-PAGE;Electrospray mass spectrometry;Molecular weight

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引用本文：

唐威华, 张景六, 王宗阳, 洪孟民. SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因[J]. 生理学报 2000; 52 (1): .

Tang Weihua, Zhang Jingliu, Wang Zongyang, Hong Mengmin. The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE. Acta Physiol Sin 2000; 52 (1): (in Chinese with English abstract).