Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin
- 作者：Kenneth V. Mills ; Katherine R. Connor ; Deirdre M. Dorval and Katherine T. Lewandowski
- 关键词：Protein purification ; Intein ; Protein splicing ; Pyrococcus abyssi
- 刊名：Analytical Biochemistry
- 出版年：2006
- 期刊代码：93_00032697
- 类别：imm
- 出版时间：1 September 2006
- 卷：356
- 期：1
- 页码：86-93
- 文件大小：304 K
摘要
The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 °C or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40 °C or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.