在上面的ReSulT中，我們介紹了BbRAG1L和bbRAG2L的特點(diǎn)和在在體轉(zhuǎn)移（ex vivo）的情況，這次我們要研究在體外（in vitro）的情況。

在這次的實(shí)驗(yàn)中，首先

We co-purified full-length bbRAG1L and bbRAG2L, each fused at its N terminus to maltose binding protein (MBP), from mammalian cells and performed cleavage reactions using substrates containing either a 5′-TIR/3′-TIR pair or a 12RSS/23RSS pair

所謂TRI pair，RSS pair就是兩個轉(zhuǎn)座子對（Figure 5B）

反應(yīng)需要鎂離子和一種人DNA結(jié)合蛋白HMGB1。可以看到bbRAG1L/bbRAG2L選手非常給力

Co-expressed bbRAG1L/bbRAG2L exhibited robust cleavage activity on the TIR substrate, with cleavage products detectable as early as 2 min.

我們想對比RAG1/2和bbRAG1L/2L的功能，于是

co-expressed RAG1/2 cleaved the RSS substrate with similar kinetics (lanes 6–10). Cleavage products had the sizes expected for double-strand breaks at the borders of the TIRs or RSSs, with both enzymes generating substantial amounts of the double-cleavage product (double asterisks). Some differences were noted in the pattern of cleavage products generated by bbRAG1L/2L as compared to RAG1/2.

那么不同在哪里？

BbRAG1L/2L generated a product corresponding to single cleavage at the 3′ TIR (asterisk), which was visible at 2 min and accumulated to high levels over the 30-min time course. In contrast, RAG1/2 generated predominantly the double-cleavage product, with single-cleavage products visible as minor species only at later time points, consistent with RAG’s well-known propensity to perform coordinated cleavage at a 12/23 RSS pair

從圖中也可以看出，RAG1/2主要產(chǎn)生兩邊的切斷，而BbRAG1L/2L主要產(chǎn)生一邊的切斷。

之后，我們開始探究BbRAG1L/2L產(chǎn)生活性的條件，我們發(fā)現(xiàn)BbRAG1L/2L兩種蛋白必須同時(shí)存在，并且突變掉D701之后，該酶也失活了。同時(shí)，該任性的酶在RSS位點(diǎn)活性非常弱。更甚于它的兄弟RAG1/2，BbRAG1L/2L更加依賴HMGB1以及Mg2+、Mn2+兩種金屬陽離子。

為了測試BbRAG1L/2L和RAG1/2是否都是相似的發(fā)夾剪切機(jī)制（圖上有）

cleavage reactions were performed using an end-labeled 3′-TIR DNA substrate, with the cleavage products analyzed by denaturing gel electrophoresis

實(shí)驗(yàn)的結(jié)果證明：確實(shí)一樣。