Anti-His-Tag Chimeric monoclonal Antibody , clone SNI02

The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403-621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1(403-621) inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416-621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1(416-621) variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1(416-621) was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1(416-621) in 7.5 M urea, different refolding conditions with varying L-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M L-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1(415-621) catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.

Immobilized metal-ion affinity chromatography (IMAC) is a valuable method for preparative and analytical-scale protein separations. Nylon 6 capillary-channeled polymer (C-CP) fibers were grafted with glycidyl methacrylate (GMA) as a monomer with ceric ammonium nitrate (in dilute nitric acid) used as the initiator. The polymerization reaction occurs rapidly (15 min) in a residential microwave. Iminodiacetic acid (IDA) is then attached to the grafted GMA polymers by reacting with the reactive terminal epoxide groups. Different parameters regarding the grafting time, initiator concentration and conversion time were investigated to find the optimal conditions for the entire modification process. The resulting nylon-IDA fibers were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM). The resulting carboxyl density and copper binding capacity were determined to be 612 +/- 21 mu mol g(-1) and 375 +/- 12 mu mol g(-1), respectively. When charged with Cu2+ ions and packed in a column format, the nylon-IDA fibers can be applied as an IMAC stationary phase for the separation of histidine rich proteins. The performance of this novel phase was evaluated through the separation of a mixture of model proteins (cytochrome C, alpha-chymotrypsinogen A and lysozyme) and a recombinant histidine-tagged protein (his-tagged ubiquitin). Despite multi-step modifications, columns of the modified fibers still maintain the anticipated high levels of throughput and efficiency, with binding capacities of 6.89 +/- 0.56 mg lysozyme g(-1) fiber and 6.32 +/- 0.12 mg His-tagged ubiquitin g(-1) fiber.