Mills Kenneth V, Connor Katherine R, Dorval Deirdre M, Lewandowski Katherine T
Department of Chemistry, College of the Holy Cross, Worcester, MA 01610, USA.
Anal Biochem. 2006 Sep 1;356(1):86-93. doi: 10.1016/j.ab.2006.04.055. Epub 2006 May 19.
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 degrees Celsius 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 degrees Celsius 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.
在深渊嗜热栖热菌中打断DNA聚合酶II DP2亚基的内含肽可以在大肠杆菌中过量表达，并作为未剪接的前体进行纯化。在37摄氏度或更高温度下进行体外孵育时，内含肽介导高效的蛋白质剪接。可以将突变引入内含肽融合蛋白中，以阻止蛋白质剪接的第二步和第三步。结果，内含肽融合蛋白可以促进N-外显肽和内含肽之间硫酯键的温度依赖性形成。这种硫酯在40摄氏度或更高温度下易受体外水解或硫解作用的影响，我们利用这种活性建立了一种温度依赖性蛋白质纯化方案。使用这种内含肽进行蛋白质纯化不需要添加外源硫醇，并且与固定化金属亲和色谱法兼容。与当前的纯化系统相比，N-外显肽C末端残基的身份对体内过早切割的切割反应影响较小，并且在体外高效切割方面与当前系统互补。
