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.