文章基本信息

标题：Purification of recombinant apolipoproteins A-I and A-IV and efficient affinity tag cleavage by tobacco etch virus protease
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作者：Matthew R. Tubb ; Loren E. Smith ; W. Sean Davidson 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2009
卷号：50
期号：7
页码：1497-1504
DOI：10.1194/jlr.D900003-JLR200
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：The expression of recombinant apolipoproteins provides experimental avenues that are not possible with plasma purified protein. The ability to specifically mutate residues or delete entire regions has proven to be a valuable tool for understanding the structure and function of apolipoproteins. A common feature of many recombinant systems is an affinity tag that allows for straightforward and high-yield purification of the target protein. A specific protease can then cleave the tag and yield the native recombinant protein. However, the application of this strategy to apolipoproteins has proven somewhat problematic because of the tendency for these highly flexible proteins to be nonspecifically cleaved at undesired sites within the native protein. Although systems have been developed using a variety of proteases, many suffer from low yield and, especially, the high cost of the enzyme.We developed a method that utilizes the tobacco etch virus protease to cleave a histidine-tag from apolipoproteins A-I and A-IV expressed in Escherichia coli . This protease can be easily and inexpensively expressed within most laboratories. We found that the protease efficiently cleaved the affinity tags from both apolipoproteins without nonspecific cleavage. All structural and functional measurements showed that the proteins were equivalent to native or previously characterized protein preparations. In addition to cost-effectiveness, advantages of the tobacco etch virus protease include a short cleavage time, low reaction temperature, and easy removal using the protease's own histidine-tag.