6xHis promotes binding of a recombinant protein to heparan sulfate.

Commercially available systems provide a variety of strategies for the expression and purification of recombinant proteins. One popular method is the polyhistidine fusion tag, which usually consists of six consecutive histidines (6×His) added to the amino or carboxyl terminus of a protein (7). The high-affinity binding of 6×His to metalchelate resins allows easy purification of 6×His-tagged proteins under both nondenaturing and denaturing conditions (6,8,15). Purified under nondenaturing conditions, 6×His-tagged proteins have been shown to maintain native conformations and biological activity (8), and the 6×His tag does not alter the activity of such recombinant proteins as endonuclease EcoRV (11) and human serum response factor (8). One producer of a 6×His-based expression system (Qiagen, Valencia, CA, USA) states in the technical manual, “the 6×His tag is very small and does not interfere with the structure or function of the recombinant protein”. However, we find that the addition of a 6×His tag to a recombinant protein increases the protein’s capacity for binding heparan sulfate. Sp17, a 17.5-kDa protein expressed on acrosome-reacted sperm, promotes sperm-egg adhesion by interacting with carbohydrates in the oocyte zona pellucida (10). In vitro analysis indicates Sp17 binds dextran sulfate and perhaps other sulfated carbohydrates such as heparan sulfate (13). We recently discovered that malignant plasma cells (myeloma cells) express Sp17 on their surface (9). To enable us to investigate the role of Sp17 in heparan sulfate-mediated cell-cell adhesion of myeloma cells, we expressed two forms of recombinant Sp17 (rSp17) with different purification tags at the N-terminus: glutathione-S-transferase (GST)-rSp17 and 6×His-rSp17. The GST tag is removed via a thrombin cleavage site allowing for purification of rSp17 with no tag, while 6×His-rSp17 retains its amino acid tag after purification. Preliminary experiments suggested that the 6×His tag interferes with the heparan sulfate-binding capacity of rSp17. Thus, to determine the extent of interference by the 6×His tag, we compared the ability of rSp17 and 6×His-rSp17 to interact with heparan sulfate. To express GST-rSp17 for the purification of rSp17 alone, the 456-bp open reading frame of human Sp17 was amplified by RT-PCR from the total RNA of a human plasma cell leukemia line, ARH-77 (3) (ATCC, Manassas, VA, USA), then ligated in-frame into the pGEX-4T-2 vector (Amersham Biosciences, Piscataway, NJ, USA) 3′ to the sequence encoding the GST gene and thrombin cleavage site. The Sp17 cDNA insert was sequenced for accuracy, and GST-rSp17 was expressed in E. coli strain BL21 (Amersham Biosciences). After purification under nondenaturing conditions using glutathione-Sepharose beads (Amersham Biosciences), rSp17 was released from the GSTrSp17-glutathione bead complex with thrombin. The thrombin was then removed from the released rSp17 using benzamidine-Sepharose (Amersham Biosciences). SDS-PAGE analysis of thrombin-released rSp17 showed one band at the appropriate size (not shown), and western analysis with Sp17 antiserum (generously provided by Dr. Michael O’Rand) confirmed the band to be rSp17 (9). To express and purify 6×His-rSp17, the cDNA of Sp17 was amplified by PCR and ligated in-frame into the pQE-30 vector (Qiagen) 3′ to the sequence encoding the 6×His tag. Before expressing the 6×His-rSp17 in E. coli strain M15, the Sp17 cDNA insert was sequenced to confirm accuracy. The 6×His-rSp17 was purified under nondenaturing conditions by affinity chroBenchmarks