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Molecular and Physiological Characterization of Two Novel Multirepeat β-Thymosins from Silkworm, Bombyx mori.

Ma S, Kang Z, Lü P, Yang Y, Yao Q, Xia H, Chen K - PLoS ONE (2015)

Bottom Line: Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period.The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection.In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, P. R. China.

ABSTRACT
β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

No MeSH data available.


Related in: MedlinePlus

The expression, purification and Western blot analysis of recombinant BmTHYs.The expressed BmTHYs proteins were purified, digested to remove GST tags and further purified and subjected to 12%SDS-PAGE (A). The prepared antiserum was used to Western blot to detect the BmTHY1 and BmTHY2 tagged with GST from the lysate of E. coli expressing the corresponding proteins (B), the BmN cell lysate (C, lane 1) and the protein extract of ovaries (C, lane 2). (A)Lane M, protein molecular weight marker; Lane 1: BmTHY1; Lane 2: BmTHY2. (B)Lane 1: GST-BmTHY1; Lane 2: GST-BmTHY2. (C)Lane M, prestained protein ladder; Lane 1: The total protein of BmN cells; Lane 2: The total protein of ovaries.
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pone.0140182.g004: The expression, purification and Western blot analysis of recombinant BmTHYs.The expressed BmTHYs proteins were purified, digested to remove GST tags and further purified and subjected to 12%SDS-PAGE (A). The prepared antiserum was used to Western blot to detect the BmTHY1 and BmTHY2 tagged with GST from the lysate of E. coli expressing the corresponding proteins (B), the BmN cell lysate (C, lane 1) and the protein extract of ovaries (C, lane 2). (A)Lane M, protein molecular weight marker; Lane 1: BmTHY1; Lane 2: BmTHY2. (B)Lane 1: GST-BmTHY1; Lane 2: GST-BmTHY2. (C)Lane M, prestained protein ladder; Lane 1: The total protein of BmN cells; Lane 2: The total protein of ovaries.

Mentions: Recombinant BmTHYs were expressed in E. coli and purified by GST affinity Chromatography (Fig 4A). As expected, the molecular weight of BmTHY1 is about 22 kDa and that of BmTHY2 is about 19 kDa, and they were expressed correctly(S1 Fig). The mouse polyclonal antiserum were prepared and successfully used to detect the GST-BmTHY1 and GST-BmTHY2, as shown by the Western blot (Fig 4B). And it is clear that this antibody could be used to investigate the protein expression profiles for BmTHYs (Fig 4C).


Molecular and Physiological Characterization of Two Novel Multirepeat β-Thymosins from Silkworm, Bombyx mori.

Ma S, Kang Z, Lü P, Yang Y, Yao Q, Xia H, Chen K - PLoS ONE (2015)

The expression, purification and Western blot analysis of recombinant BmTHYs.The expressed BmTHYs proteins were purified, digested to remove GST tags and further purified and subjected to 12%SDS-PAGE (A). The prepared antiserum was used to Western blot to detect the BmTHY1 and BmTHY2 tagged with GST from the lysate of E. coli expressing the corresponding proteins (B), the BmN cell lysate (C, lane 1) and the protein extract of ovaries (C, lane 2). (A)Lane M, protein molecular weight marker; Lane 1: BmTHY1; Lane 2: BmTHY2. (B)Lane 1: GST-BmTHY1; Lane 2: GST-BmTHY2. (C)Lane M, prestained protein ladder; Lane 1: The total protein of BmN cells; Lane 2: The total protein of ovaries.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608725&req=5

pone.0140182.g004: The expression, purification and Western blot analysis of recombinant BmTHYs.The expressed BmTHYs proteins were purified, digested to remove GST tags and further purified and subjected to 12%SDS-PAGE (A). The prepared antiserum was used to Western blot to detect the BmTHY1 and BmTHY2 tagged with GST from the lysate of E. coli expressing the corresponding proteins (B), the BmN cell lysate (C, lane 1) and the protein extract of ovaries (C, lane 2). (A)Lane M, protein molecular weight marker; Lane 1: BmTHY1; Lane 2: BmTHY2. (B)Lane 1: GST-BmTHY1; Lane 2: GST-BmTHY2. (C)Lane M, prestained protein ladder; Lane 1: The total protein of BmN cells; Lane 2: The total protein of ovaries.
Mentions: Recombinant BmTHYs were expressed in E. coli and purified by GST affinity Chromatography (Fig 4A). As expected, the molecular weight of BmTHY1 is about 22 kDa and that of BmTHY2 is about 19 kDa, and they were expressed correctly(S1 Fig). The mouse polyclonal antiserum were prepared and successfully used to detect the GST-BmTHY1 and GST-BmTHY2, as shown by the Western blot (Fig 4B). And it is clear that this antibody could be used to investigate the protein expression profiles for BmTHYs (Fig 4C).

Bottom Line: Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period.The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection.In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, P. R. China.

ABSTRACT
β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

No MeSH data available.


Related in: MedlinePlus