Introduction

Rice feeds more than one-third of the global population and is an important model cereal crop due to its 389-Mb genome [1] and convenient gene transfer methods [2]. About 45,000 protein-coding sequences have been predicted in rice haploid genome

[3]. Studying the functions of rice genes is an active research area using primarily genetic methods. An additional area of research is discovering protein interactions, because they can reveal some insights into a protein's function and in vivo mechanisms

[4]. A recently developed TAP-tag technology is a state-of-the-art methodological approach [5] for large-scale analyses of protein complexes in vivo [6, 7] and is comparatively easy for researchers with a limited experience in protein purifications. TAP-tag technology has been successfully used in yeast [6], Drosophila [8], mammalian cells [9], E. coli [10], Chlamydomonas [11], and higher plants [12-15]. In TAP the protein complex is sequentially affinity purified, and the proteins present in the complex are identified with the help of MS and bioinformatics.

The TAP-tag was first used in yeast [16] for large-scale PPI studies. The first successful report on the use of TAP-tag technology for the purification of a protein complex in planta was demonstrated by our group [12]. The TAP-tag can be fused on either C- or N-terminus to the protein of interest and are available in vectors incorporating GATEWAYTM (Invitrogen) recombination sites for expression of fusion proteins under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The usefulness of the TAP-tag is further demonstrated by analyzing over 100 TAP-purified-protein kinase purifications [13, 15] in our lab.

There are several other versions of TAP tags available. Among these, ProA-3C protease-6xHis:9xmyc [14], BirA (biotin):target protein:TAP-tag [17], ProA-TEV-FLAG [9], and CBP:6xHis:3xHA [18] are available from academic institutions, while InterPlay™ TAP System (SBP:CBP) (Stratagene) and FLAG®HA TAP kit (Sigma) are distributed by companies. In a complementation study of mutant lines with seven TAP-tagged proteins, it was shown that three did not complement, two partially complemented, and two fully complemented the mutant phenotypes [14]. This indicates that the expression and function of TAP-tagged proteins are specific to each fused protein. The probability of success with the method will be discussed more fully below.

36.2 METHODOLGY AND STRATEGY TAP Methodology Overview

A TAP project starts with making a gene construct that fuses the TAP-tag to a target protein of interest. This construct is then transformed into the plant, and transgenic plants expressing the fusion protein are identified. Transient expression is readily detectable in N. benthamiana agroinfection assays [19] and can be useful as a quick check that the protein fusion is expressed and functional. Crude protein extracts from the initial transgenic plant, or more typically the progeny plants, are prepared from these transgenic cells. The fusion protein as well as interacting proteins present in a protein complex are then recovered by TAP. The TAP method involves two sequential affinity purification steps: an IgG column followed by CaM column purification. The two purification steps are followed by separation of the individual purified proteins by SDS-PAGE. The protein bands are visualized with a fluorescent dye, excised, and analyzed by MS to identify the proteins. The TAP strategy has been summarized in Figure 36.1.

TAP-Tag Structure

Typically, a TAP-tag consists of two protein-binding domains separated by a protease cleavage site. The Protein A domain from S. aureus (ProA) and CaM binding protein (CBP) domain in the TAP-tag have sufficient affinity for efficient recovery of a fusion protein present at a low level in a complex crude extract from a transgenic plant (http://www-db.embl-heidelberg.de/jss/servlet/de.embl.bk.wwwTools.GroupLeft EMBL/ ExternalInfo/seraphin/TAP.html). These two affinity tags, ProA and CBP, are separated by a TEV protease cleavage site. TEV cleavage is used to separate the ProA domain, which remains tightly bound to the IgG beads, from the released protein complex. The recombinant TEV protease recognizes the ENLYFQG protein

300 ml crude extract from 60-70 grams of rice leaves in six 50 ml centrifuge tubes; each tube having 200 pi IgG beads I ~

IgG binding at 4°C on a rotator

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