Department of Biochemistry, University of Washington, Seattle, WA, USA.
BMC Biotechnol. 2011 Jun 27;11:71. doi: 10.1186/1472-6750-11-71.
Inteins are proteins that catalyze their own removal from within larger precursor proteins. In the process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity are independent and distinct domains in the folded structure. We show here that other biochemical activities can be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-terminal extein to create a cassette to introduce GFP within the interior of a targeted protein.
The Pch PRP8 mini-intein of Penicillium chrysogenum was modified to include: 1) aminoglycoside phosphotransferase; 2) imidazoleglycerol-phosphate dehydratase, His5 from S. pombe ; 3) hygromycin B phosphotransferase; and 4) the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost endonuclease. When expressed in E. coli, all of the modified inteins spliced at high efficiency. Splicing efficiency was also greater than 96% when expressed from a plasmid in S. cerevisiae. In addition the inteins conferred either G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast genetic background. DNA encoding the marked inteins coupled to GFP as the N-terminal extein was PCR amplified with ends homologous to an internal site in the yeast calmodulin gene CMD1. The DNA was transformed into yeast and integrants obtained by direct selection for the intein's marker. The His5-marked intein yielded a fully functional calmodulin that was tagged with GFP within its central linker.
Inteins continue to show their flexibility as tools in molecular biology. The Pch PRP8 intein can successfully tolerate a variety of genetic markers and still retain high splicing efficiency. We have shown that a genetically marked intein can be used to insert GFP in one-step within a target protein in vivo.
内肽酶是一种能够催化自身从较大的前体蛋白中去除的蛋白质。在这个过程中，它们将侧翼的蛋白质序列（称为 N 和 C 末端外显子）拼接在一起。大型内肽酶通常具有一种参与维持宿主中内肽酶的同源内切核酸酶。剪接和核酸酶活性是折叠结构中独立且不同的结构域。我们在这里表明，可以在不影响剪接的情况下，将其他生化活性取代内切核酸酶插入到内肽酶中，并且这些活性可以为内肽酶提供遗传选择。我们已经将这样一个遗传标记的内肽酶与 GFP 作为 N 末端外显子偶联，以创建一个盒，将 GFP 引入靶向蛋白的内部。
青霉属内切酶 PRP8 的 mini-intein 被修饰以包括：1）氨基糖苷磷酸转移酶；2）咪唑甘油磷酸脱水酶，来自 S. pombe 的 His5；3）潮霉素 B 磷酸转移酶；和 4）转录激活因子 LexA-VP16。这些蛋白质被插入到丢失的内切核酸酶的位置。当在大肠杆菌中表达时，所有修饰的内肽酶都以高效率剪接。当在酿酒酵母中从质粒表达时，剪接效率也大于 96%。此外，根据插入的标记和酵母遗传背景，内肽酶赋予 G418 或潮霉素抗性，或组氨酸或亮氨酸原代表型。带有 GFP 作为 N 末端外显子的标记内肽酶的 DNA 用与酵母钙调蛋白基因 CMD1 内部位点同源的末端进行 PCR 扩增。DNA 转化到酵母中，并通过直接选择内肽酶的标记获得整合子。His5 标记的内肽酶产生了一种完全功能的钙调蛋白，其在中央接头内被 GFP 标记。
内肽酶继续作为分子生物学工具展示其灵活性。Pch PRP8 内肽酶可以成功耐受多种遗传标记，同时仍保持高剪接效率。我们已经表明，一个遗传标记的内肽酶可以用于在体内一步将 GFP 插入到靶蛋白中。
