New England Biolab

New England Biolab’s IMPACT System
The IMPACT-CN (Intein Mediated Purification with an Affinity Chitin-binding Tag) System from New England Biolabs is a novel protein purification system which utilizes the inducible self-cleavage activity of a protein splicing element (termed intein) to separate the target protein from the affinity tag. It distinguishes itself from all other purification systems by its ability to purify, in a single chromagraphic step, a native recombinant protein without the use of a protease. The concept of IMPACT purification has evolved from studies of protein splicing mechanisms. A target protein is fused to a self-cleavable intein tag in which a chitin binding domain allows affinity purification of the fusion precursor on a chitin column. In the presence of DTT, the intein undergoes specific self-cleavage which releases the target protein from the chitin bound intein tag resulting in a single column purification of the target protein. The IMPACT-CN System contains expression vectors which allow the fusion of the cleavable intein tag to either the C-terminus or N-terminus of a target protein. The pTYB vectors use a T7 promoter-driven system to achieve high levels of expression and tight transcriptional control in E. coli. Syphilis is a disease caused by the organism Treponema Pallidum (sometimes referred to as TP). The diagnosis of syphilis is made by using an immunoassay to detect anti-TP antibodies in the serum or plasma. The surface of TP has a large number of membrane antigens and the immunoassay utilizes the antigen-antibody reaction between the membrane antigen and the anti-TP antibody in the blood specimen. Thus having purified recombinant TP antigens is the best way to get the most specific and sensitive results. We have used this kit to express and purify the TP membrane protein TP-17, which has been reported to be one of the more antigenic surface proteins. We chose pTYB2 vector to clone the TP-17 gene; and transformed the plasmid into the E. coli host strain ER 2566 that carries the T7 RNA Polymerase gene. The cells were grown at 37°C in LB medium containing 100 μg/ml ampicillin. When the OD600 of the culture reached 0.6, protein expression was induced at 20°C with IPTG (final concentration of 0.5 mM). The cells were then harvested and crude extract was isolated by sonication on ice. After equilibrating the chitin column, the clarified extracts were loaded at a rate no faster than 0.5 ml/min. Thus the fusion protein, which contained the TP-17 protein with the intein tag (about 62 KDa total) is now bound to the column matrix. The column was washed with 20 times of column buffer at a flow rate of under 1ml/min to ensure that all traces of crude extract have been washed off the sides of the column. The column was flushed quickly with about 3 column volumes of 50 mM DTT (freshly diluted in Cleavage Buffer or other suitable buffer) to evenly distribute DTT throughout the column before incubating at 4°C overnight. After the overnight incubation, the target protein was eluted using additional cleavage buffer without DTT. Then we get the purified recombinant TP-17 protein, which is about just 17 KDa. Using this affinity purified TP-17 antigen, we have developed a reliable ELISA method to detect Anti-TP antibodies in human blood specimens to diagnose Syphilis.。