The Identification, Expression And Function Analysis Of Hypothetical Proteins Which Have A DUF985 Motif And A Phd-like-VIAF Motif From Amphioxus (Branchiostoma Belcheri)

The progress in genome sequencing has led to an increasing submission of uncharacterized hypothetical genes with the domain DUF985 in GenBank, and none of these genes is related to a known protein or a protein of known function. In this study we have shown conclusively that the DUF985-containing hypothetical gene from amphioxus Branchiostoma belcheri, BbDUF985, encodes a novel member of phosphoglucose imomerase (PGI). BbDUF985 is expressed in vitro in both Escherichia coli and Pichia pastoris, and the specific activity of the recombinant protein expressed in the eukaryotic system is approximately 440 U/mg of protein, which is significantly higher than that of BbDUF985 expressed in the prokaryotic system. Tissue-section in situ hybridization as well as immunohistochemistry demonstrate that BbDUF985 is expressed in vivo in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In the CHO cells transfected with the expression plasmid pEGFP-N1/BbDUF985, the fusion protein is targeted in the cytoplasm of CHO cells, suggesting that BbDUF985 is a cytosolic protein. On the other hand, western blotting indicates that BbDUF985 can also be secreted into the humoral fluids of amphioxus. The presence of both cytosolic and extracellular BbDUF985 suggests it may be like PGI playing a multiple function. These are the first report describing the successful expression, biochemical characterization and cellular localization of a hypothetical protein with the domain DUF985, providing a framework for further understanding the biochemical properties and physiological function of DUF985-domain-containing hypothetical proteins.We also report the identification of a hypothetical gene with the conserved motif Phd-like-VIAF from amphioxus Branchiostoma belcheri. Many genes with the domain Phd-like-VIAF have been deposited in GenBank, and these genes are related to a known protein, phosducin-like 3, their function remains uncertain. In this study Amphi-phdL is expressed in vitro in Escherichia coli. Tissue-section in situ hybridization as well as immunohistochemistry demonstrate that Amphi-phdL is expressed in vivo in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In the CHO cells transfected with the expression plasmid pEGFP-N1/Amphi-phdL, the fusion protein is targeted in the cytoplasm of CHO cells, suggesting that Amphi-phdL is a cytosolic protein. Western blotting indicates that Amphi-phdL is present in both tissue extract and humoral fluids of amphioxus. The study of function of Amphi-phdL is now under way.