Fibroblast growth factor 19 (FGF19) is the human ortholog of mouse

Fibroblast growth factor 19 (FGF19) is the human ortholog of mouse FGF15 and both proteins function as an endocrine signal to regulate numerous liver functions. activities confirmed by suppressing hepatic Cyp7a1 gene transcription in mice and by activating ERK1/2 signaling pathway in HepG2 cells. In contrast soluble FGF15 protein in cytoplasm remained very PHA-739358 low using these strategies. In summary we have successfully developed a method to express functional FGF19 protein in prokaryotic cells and this strategy may be adapted for the expression of other disulfide-containing proteins. Introduction Fibroblast growth factor 19 (FGF19) is usually expressed in human liver and intestine and shows different tissue distribution from its mouse ortholog FGF15 which is only expressed in the intestine [1]. However both proteins function as enterohepatic hormones and they are secreted from the small intestine to regulate bile acid homeostasis in the liver. After being secreted into the portal blood circulation FGF15/19 binds to its receptor FGFR4 in the liver [2] [3] and activates downstream signaling pathways to suppress the transcription of the gene encoding cholesterol 7α-hydroxylase (Cyp7a1) the rate-limiting enzyme for bile acid synthesis [3]-[5]. Moreover FGF15/19 has been shown to promote liver tumorigenesis [6] energy metabolism [7] insulin sensitivity [8] and liver regeneration [9] but the underlying mechanisms Rabbit polyclonal to COT.This gene was identified by its oncogenic transforming activity in cells.The encoded protein is a member of the serine/threonine protein kinase family.This kinase can activate both the MAP kinase and JNK kinase pathways.. have not been fully clarified. Pure and functional proteins produced by an efficient method can provide a valuable tool to greatly improve the research of FGF19/15. Due to easy handling inexpensive cultivation and large-scale production the bacterial system is a popular and well characterized prokaryotic host system for heterologous protein expression [10] [11]. However the system also contains a few limitations and expression of eukaryotic proteins in a bacterial system has been usually challenging especially when these proteins contain disulfide bonds [12] [13]. Disulfide bonds are very common in mammalian proteins and are crucial for proper protein folding stability and activity. They are created into the covalent bond by the oxidation of thiol groups between two cysteine residues in the protein. Cytoplasm of is constantly maintained as a reducing environment therefore in general the cytoplasm is not favorable for the expression of proteins made up of disulfide bonds and the formation of disulfide-bond containing proteins in bacterial cytosol is usually unstable and PHA-739358 normally forms inactive inclusion bodies. Therefore additional refolding is required to obtain biologically functional proteins. However it is well known that refolding of protein in inclusion body is often unpredictable and challenging PHA-739358 [13]-[17] in addition to being time consuming and requiring a large amount of reagents. Overall generation of protein in soluble form is the favored choice. Extensive efforts have been made to overcome these obstacles to improve soluble expression of different disulfide-bonded proteins in the cytosol of cytoplasm are actively PHA-739358 kept reduced by pathways including thioredoxin reductase and glutaredoxin [18]-[20] so one strategy is to alter these reducing pathways to change the cytoplasmic thiol-redox equilibrium environment. There are various types of commercially available mutant strains (AD494 Origami (Novagen) SHuffle (New England Biolabs) [18] [19] [21] which lack thioredoxin reductase (Δmutant strains and soluble protein expression in bacteria cytoplasm were decided. Materials and Methods Ethics Statement Mice were bred and managed in the facility of the Laboratory of Animal Research at the University or college of Kansas Medical Center and were housed in rooms under a standard 12-hr light/dark cycle with access to chow and water ad libitum. All protocols and procedures were approved by the Animal Care and Facilities Committee (ACFC) at the Rutgers University PHA-739358 or college The State University or college of New Jersey and are in accordance with the NIH PHA-739358 and AALAC Guidelines (protocol.