Introduction

Three aspects of intein biology suggest a wide variety of uses in biotechnology. The first, described in this volume by Dassa and Pietrokovski, is that interns are genetically mobile, and a characteristic exhibited by many inteins is the retention of splicing activity when artificially moved to foreign contexts. The second is that inteins can be used to reversibly inactivate non-native host proteins through internal and external gene fusions, allowing inteins to be used as molecular triggers in various reporter systems. This aspect of intein biology forms the foundation for this chapter and the systems described within. Finally, as explained by Mills and Paulus in this volume, the splicing reaction itself is fairly well understood and several intein structures have been solved (see Moure and Quiocho, this Vol.). This has allowed inteins to be modified for new biological applications, including those described in chapters by Chong and Xu; Tavassoli, Naumann and Benkovic; Ozawa and Umezawa; and Wood, Harcum and Belfort. The repertoire of available inteins has recently been augmented with examples of split inteins that can assemble and splice in trans, enabling the development of additional strategies for gene assembly and activation.

The ability of inteins to function in non-native contexts initially facilitated the development of several convenient model systems for intein study and characterization (Davis et al. 1991; Cooper et al. 1993; Xu et al. 1993; Chong et al. 1996). In time, however, it became clear that the flexibility and robustness of inteins suggested a wide variety of potential applications (Perler and Adam 2000). In pursuit of these applications, a number of additional systems have been developed to link intein activity to readily detectable or selectable

D.W. Wood (e-mail: [email protected]), G. Skretas Department of Chemical Engineering, Princeton University, Princeton, New Jersey, USA

Nucleic Acids and Molecular Biology, Vol. 16 Marlene Belfort et al. (Eds.) Homing Endonucleases and Inteins © Springer-Verlag Berlin Heidelberg 2005

phenotypes in expressing cells. In ail cases, these systems are based on the genetic fusion of an intein to a well-characterized reporter protein. Splicing, or in some cases cleaving by the intein, activates the reporter protein, allowing the easy identification of conditions that affect the activity of the intein (Fig. 1).

Applications of these systems can be broken down into four broad categories. The first is simply to provide a tool to study intein activity under various conditions. Several early reporters provided initial insights into intein function in non-native host cells and proteins (Davis et al. 1992; Cooper et al. 1993), while more recent reporters have been used to examine the effects of specif-

Fig. 1. Reporter and selection strategies as described in this chapter. Intein splicing or cleaving activates a reporter protein, leading to a change in growth phenotype (selection), or the presence of an assayable activity or change in molecular weight of the precursor (screening). Reporter proteins are designated for each strategy as discussed in the text.

Fig. 1. Reporter and selection strategies as described in this chapter. Intein splicing or cleaving activates a reporter protein, leading to a change in growth phenotype (selection), or the presence of an assayable activity or change in molecular weight of the precursor (screening). Reporter proteins are designated for each strategy as discussed in the text.

ic intein and extein mutations on intein function (Xu et al. 1993; Chong et al. 1996; Xu and Perler 1996; Derbyshire et al. 1997; Kawasaki et al. 1997; Ghosh et al. 2001; Lew and Paulus 2002). A second application has been to use intein fusions to select open reading frames (ORFs) from large DNA libraries. In this case, the intein is linked to a selectable marker and the DNA library is inserted in such a way that non-ORF sequences will disrupt intein expression and function (Daugelat and Jacobs 1999). A third use for intein-reporter fusions is to facilitate the rapid screening of large chemical libraries for compounds that inhibit or otherwise affect intein splicing (Adam and Perler 2002; Lew and Paulus 2002; Gangopadhyay et al. 2003; Cann et al. 2004). Although there are many possible uses for such compounds, an important initial purpose for them would be to provide a new class of antibiotics against pathogenic intein-harboring bacteria (Belfort 1998; Paulus 2003; Liu and Yang 2004). Finally, an important application for many of these systems has been the rapid isolation of valuable intein mutants through directed evolution. Intein variants generated by this method have been successfully used for protein purification (Wood et al. 1999), and have shown potential as a general means to post-translation-ally activate arbitrary target proteins in vivo on demand (Buskirk et al. 2004; Zeidler et al. 2004; Skretas and Wood 2005).

This chapter is devoted to describing the development and use of reporter systems for intein splicing and cleaving. For each method, the underlying principle will be described, along with its most prevalent applications and significant results. The variety of these systems is increasing rapidly, and it is becoming clear that virtually any genetic selection scheme can be modified to include an intein-splicing component.

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