analysis at 25 kDa (LvDBP23-His) with anti-His antibody. The result shows the recombinant protein is presented in medium and cell fraction. The recombinant proteins apparently are made of two bands (Fig. 3). The sf9 cell expression system used in the study produce secretary recombinant proteins. The cell extracts have two bands with more the large-size protein while the extracts from the medium produce two bands with more the small-size proteins. Therefore, we believe the two bands are the large-size protein with the signal peptide predominantly in cell extract, and the small-size protein without the signal peptide predominantly in the medium. The protein levels appear to increase dramatically from 0 to 72 hrs of cell culture (Fig. 3). Expression of LvDBP23 gene in shrimp tissues To verify the expression of LvDBP23 transcripts and protein, we analyze the mRNA levels of LvDBP23 in various tissues from juvenile shrimp by reverse transcriptase-PCR. As shown in Fig. 4, the mRNA expressions were detected in intestine (I), lymphoid (L), gill (G), abdominal muscle (AM) and swimming leg muscle (SM) with the highest level in abdominal muscle. We also performed the protein expression by western blot analysis. The cytosolic and nuclear proteins were extracted and detected with LvDBP23 polyclonal antibody, which was customer-produced by Genscript. LvDBP23 protein was detected in the cytosolic fraction of the swimming leg muscle tissue (SM) at the molecular size 23 kDa (Fig. 5). These results suggested that the LvDBP23 is not a muscle- specific protein, but expressed in several other types of shrimp tissues. LvDBP23 protein was not detected in AM and other tissue samples. One possible reason may be due to the protein composition in AM samples, which may have a low percentage of the target protein (LvDBP23), but a large percentages of structural proteins such as actin, myosin etc. it may also be caused by a high degree of post-translational modifications in the tissues used in the Western blot analysis. The exact reasons of undetectable levels of LvDBP23 protein in these tissues deserve further investigations. LvDBP23 expression during molting cycle The LvDBP23 gene was originally identified from shrimp muscle cDNA library and had the highest expression in abdominal muscle tissue. We performed RT-PCR to study LvDBP23 gene expression in shrimp abdominal muscle during different stages of molting cycle. The result showed that LvDBP23 expression level was highly up-regulated in the intermolt stage (Fig. 6). Earlier reports indicate Figure 3. LvDBP23 protein expressions in Sf9 insect cell. The plasmids pIEx-5- LvDBP23-His was transfected to Sf9 insect cells. Transfected cells were incubated at 28uC for 0, 24, 48 and 72 hrs. Expressions of LvDBP23 were detected by Western blot analysis with anti-His.tag primary antibody. Lane M0, C0, M24, C24, M48, C48, M72 and C72 represent the protein extracts isolated from medium (M) or cellular (C) extracts of the indicated time of cell culture. doi:10.1371/journal.pone.0019959.g003 A Novel Shrimp DNA-Binding Protein LvDBP23 that there is high muscle protein synthesis during intermolt [5,9]. A high level of LvDBP23 in the intermolt stage suggests that LvDBP23 may involve in the regulation of shrimp muscle protein synthesis. Net muscle gain during a molt cycle is the result of protein synthesis and degradation. Although factors that control and influence both protein synthesis and degradation are critical for understanding shrimp growth, there is very limited research data available for shrimp species. The muscle morphology and biochemical changes of L. vannamei during molting process have been studied by Yang and his research group [4,10]. They found that the muscle structural a-actin and cytoskeleton b-actin were increased during the intermolt stages, suggesting the high muscle growth during these stages. Another protein named LvSUMO-1 has been reported as the regulatory protein in shrimp myogenic differentiation and muscle formation [11]. We have not specifically identified muscle regulatory proteins well studied in vertebrate animals from this shrimp cDNA library. Several myogenic regulatory factor (MRF) proteins expressed during myoblast cell differentiation such as MyoD, myogenin, Myf5, and MRF4 have also been well characterized in model animals [2]. The MyoD gene family has been shown to be conserved from Drosophila to vertebrates. In the invertebrates, one type of MRF family members was identified in the species C. elegans, sea urchin and Drosophila. The nautilus protein (NAU) was identified in Drosophila, it has a typical feature of MRF protein with a bHLH domain and has 90% similarity to the vertebrate MRF members [12]. LvDBP23 is not grouped to any these muscle regulatory proteins or protein family. In vitro nucleic acid-binding assays of LvDBP23 Different amounts of total secreted recombinant protein of GFP-His (control) and LvDBP23-His were incubated with calf thymus ssDNA-cellulose and dsDNA-cellulose beads. The result showed that only LvDBP23 protein could bind with both ssDNA and dsDNA (Fig. 7). The binding efficiency was highly dependent on the amount of protein. LvDBP23 protein showed higher interaction with ssDNA than dsDNA (Fig. 7A). In vitro nucleic acid-binding assay had been used to identify DNA/RNA binding proteins such as GR-RBP4 , DdrB, ribonucleoprotein and SSB [13–16]. Based on the earlier Gly-rich domain prediction (94–130 amino acid), the results from DNA binding assays clearly suggest that LvDBP23 is a putative DNA-binding protein. According to the mRNA expression of LvDBP23 during molt cycle clearly showed the expression only in intermolt, whose stage is characterized by high muscle protein synthesis [9,12]. This may indicate that LvDBP23 may act as transcription factor regulating the muscle protein gene expression. Further experiments in identifying the specific DNA sequence with which LvDBP23 interacts will help classify its protein nature and function. Several transcription factors have been reported to consist of Gly-rich amino acid region. For example, transcription factor NF- kB is originally identified as a transcription factor that binds to the kB site in the intronic enhancer of the immunoglobulin k light- chain gene in B lymphocytes [17]. The Gly-rich region in NF-kB p105 functions as a processing signal for the generation of the p50 subunit [18]. In Drosophila, a DNA binding protein named MSL1 also contains a glycine-rich motif between the basic and leucine- zipper-like motifs, which mediates MSL1 self-association in vitro and binding of the amino-terminal region of MSL1 to the MSL complex assembled on the male X chromosome. Its basic region may mediate DNA binding, and the glycine-rich region may promote the association of MSL1 complexes to closely adjacent sites on the X chromosome [19]. However, glycine-rich proteins in PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e19959