Phosphorylation Of Bcl10 In Immune Cell Receptor Signaling & Use Of Ssp DnaB Mini-intein As A Fusion Partner For Preparation Of Recombinant Human Brain Natriuretic Peptide

Phosphorylation of Bcl10 In Immune Cell Receptor SignalingBel10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase activation and recruitment domain (CARD) and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-κB activation and lymphocyte development and functions. Our cunent study has discovered that T cell activation induced phosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the TCR-induced phosphorylation sites. Substitution of S138 to alanine residue impaired T cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-κB activation and enhancement of IL-2 production. These findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T cell receptor-mediated NF-κB activation. We have also found hyperphosphorylation of Bcl10 enriched in membrane following B cell activation, which might be another regulatory forms of Bcl10 to immune cells.Use of Ssp DnaB Minl-Intein as a Fusion Partner for Preparation of Recomblnant Human Brain Natriuretic PeptideSome recent progress in the studies of the structure and function of the intein as well as the mechanism of intein-mediated protein splicing and its application were described. Currently, the intein was widely used for protein purification, protein ligation, the preparation of cyclic peptides and toxic proteins, which suggested that intein was much valuable for the exploration of the relationship between structure and function of a protein. Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94×10^-6...