SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因

Ｈｉｓｔａｇ／ＮｉＮＴＡ系统是新发展起来的一个亲和纯化重组蛋白的有用工具，现常用于基因编码产物的特性研究中。ＳＤＳＰＡＧＥ是实验室测定蛋白质分子量通常采用的方法，而许多实验室用此方法检测Ｈｉｓｔａｇ融合蛋白时却常发现测得的分子量偏大，产生偏差的原因尚未阐明。为弄清这一问题，本实验室在研究一个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。

The Cause of Deviation Made in Determining the Molecular Weight of His-tag Fusion Proteins by SDS-PAGE

The His tag/Ni NTA system is a useful tool for affinity purification of recombinant protein. It is currently used in characterization of gene products. SDS polyacrylamide gel electrophoresis is a method commonly used in laboratory to determine the molecular weights of proteins. It has been reported that deviation is often made in determining the molecular weight of His tag fusion protein by SDS PAGE, however the causes leading to this deviation is not clear yet. 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 (Fig.1). The results of C terminal amino acid sequencing (Fig.2) and electrospray mass spectrometry analysis of this protein (Fig.3) 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 (Fig.4). 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 researchers using His tag fusion protein.