National College of Singapore

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Effective and Fast Affinity Purification of Proteins Utilizing Recombinant Fusion Proteases
AbstractIn the affinity purification of recombinant fusion proteins, the ratelimiting step is generally the effective proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a fast, convenient and efficient method of affinity purification which can conquer this limitation. In 1 example of the method,replica chanel handbags, the protease 3C from a picornavirus (3Cpro), which cleaves particular sequences containing a minimum of six amino acids, has been expressed as a fusion with glutathione Stransferase. The resultant recombinant 'fusion protease' cleaves fusion proteins bearing (from the aminoterminus) the exact same affinity tail as the fusion protease, a 3Cpro cleavage recognition website, and the recombinant protein of curiosity. The recombinant protein is purified in a single chromatographic stage which eliminates each the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in 1 step, and the option of including any preferred amount of fusion protease to ensure efficient cleavage. The possible versatility of the technique is proven by the use of numerous affinity tails and option fusion proteases.
1Protein Engineering Laboratory, Institute of Molecular and Mobile Biology, National University of Singapore, ten Kent Ridge Crescent, Singapore 0511, Republic of Singapore.
2Papillomavirus Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, ten Kent Ridge Crescent, Singapore 0511, Republic of Singapore.
3Neuropeptide Laboratory, Institute of Molecular and Mobile Biology, National University of Singapore,chanel outlet store, 10 Kent Ridge Crescent, Singapore 0511,replica chanel, Republic of Singapore.
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