Increased Expression Purification And Characterization Of The Ww Domain Of Ca150fbp28

Yusuke Kato, Yoriko Sawano and Masaru Tanokura

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Abstract: WW domain is well known protein module that mediates protein through protein interactions by binding to proline-containing ligands. Based on the ligand predilections, WW domains have been classified into three major groups, I, II/III and IV. Group-II/III WW domains have been reported to bind proline-rich ligands.

CA150/FBP28 is one of splicing factors, which enhances the efficiency of splicing and maturation of mRNA (Lin et al., 2004). The WW domain of CA150/FBP28 belongs to Group-II/III and was shown to have binding specificity to the -Pro-Pro-Leu-Pro- sequence. However, the binding is quite subtle and the quantitative analysis of the binding is required to confirm and evaluate the binding. Many WW domains showed relatively weak binding (Kato et al., 2004). Quantitative assay such as surface plasmon resonance or isothermal titration calorimetry needs large amount of protein samples (approximately tens of milligram per each sample) especially in the cases of the analysis in weak binding conditions.

The expression system of WW domains is mainly based on the GST-fusion system. However, the amount of the production was not enough with the conventional shaking cultivation. Thus, instead of shaking, we utilized an air pump to cultivate Ecoli to produce more GST-fused WW domain. In the case of the WW domain of CA150/FBP28, apparently increased production of the fusion protein was observed with this method. In addition, the amount of culture liquid per one flask can be increased because the intense agitation is not needed.

The GST-fused WW domain was successfully purified using glutathione sepharose. Then, the fusion protein was digested with precession protease. GST forms dimer, which can interfere the accurate analysis of the binding property of the WW domain due to artificial avidity. The WW domain is isolated from GST with reverse-phase chromatography. The purity of the protein is over 95% with the analysis of mass spectrometry. The WW domain of CA150/FBP28 showed to have the activity to bind to its ligand

containing -Pro-Pro-Leu-Pro- sequence. The binding strength was approximately 1mM, which indicates avidity-free binding because the strength was rather weak compared with other WW domains.

This production system is easily applicable to other recombinant proteins including WW domains.

Key words: WW domain, surface plasmon resonance, protein expression

1. INTRODUCTION

CA150/FBP28 interacts with the PL motifs (-Pro-Pro-Leu-Pro-) or other Proline-rich sequences of other cytoplasmic proteins via its WW domains. The second WW domain of CA150/FBP28 was shown to bind the PL motif of formin1. Recent report showed that the second and third WW domain of the CA150/FBP28 concern the activation of pre-mRNA splicing2. CA150/FBP28 is dispensable in this process but activates and enhances the maturation of pre-mRNA to mRNA.

WW domain forms a quite small protein module that is constituted of approximately 30 amino acid residues. The domain is one of smallest protein modules that have no disulfide bonding or additional cofactors. Thus, the folding is ruled only the hydrogen bonding and Van del Waals force.

Despite such small molecular size, WW domains play roles in a diverse variety of cell processes, such as cell cycle regulation, ubiquitin ligation, transactivation and other cytoplasmic signal transduction. The genetic diseases that WW domains concern were demonstrated in Duchenne/Becker muscular dystrophy, Liddles syndrome, Huntingtin disease and Alzheimer's disease.

Former, the molecular configuration and other interaction study showed that WW domains were grouped into at least four groups. The Group I and IV identify the PY motif (-Pro-Pro-Xxx-Tyr-) and the pS/pT-P (-phospho Ser/phospho Thr-Pro- sequence), respectively3. Group II and III were considered to bind to the PL (-Pro-Pro-Leu-Pro-) and PR (Pro-rich sequence with Arg) motifs, respectively. But, the grouping of type II and III was confused due to its ambiguous ligand specificity. Thus, we have been demonstrating the nature of WW domains of this category. As a result we have proposed a new grouping method of WW domains, the three grouping model4. In this model, we do not distinguish the Group II and III because the WW domains of Group II and III can bind the ligands for both groups. The binding specificity of those WW domains has some predilection for those ligands but do not show any exclusiveness. In addition, the modeled structures of

Group II/III showed common surface structure, which is used to recognize those ligands.

At present, to elucidate detailed mechanism of ligand recognition of Group II/III is most pursued in terms of its characterization because the ligand-domain complex structure of this category has never been reported. Meanwhile, the importance of this category is progressively growing because the recent studies of Fe65 or FCA showed the indispensable role of WW domains in the regulation of transactivation or flowering timing control5,6.

Thus, future aim of our present study is to obtain more robust evidence for the ligand recognition of Group II/III WW domains. For the breakthrough of this state of confusion, we need more detailed and exhaustive assay of WW domains such as structural, proteomic and quantitative analysis for the binding property of the category. Our present study shows the technical basis of our strategy, which is constituted of the efficient production and purification of WW domains and the quantitative assay of the binding.

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