Molecular cloning and expression analysis of porcine gamma-interferon-inducible lysosomal thiol reductase (GILT).

A porcine interferon-gamma-inducible lysosomal thiol reductase (GILT) cDNA, designated pGILT, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pGILT consists of 1,062 bp with a 741 bp open reading frame, encoding 246 amino acids, with a putative molecular weight of 29.5 kDa.

Expression, purification and characterization of anti-BAFF antibody secreted from the yeast Pichia pastoris.

B-Cell activating factor (BAFF) is critical for B cell survival and maturation; excessive expression of it corrupts B-cell tolerance and may lead to autoimmunity. The gene, scFv-Fc, coding for the antibody of BAFF was inserted into the eukaryotic expression vector, pPICZalphaA, and transformed into Pichia pastoris. A high-level expression strain was obtained using a 'yeastern blotting' method.

Molecular cloning and expression analysis of interferon-gamma-inducible-lysosomal thiol reductase gene in orange-spotted grouper, Epinephelus coioides.

In mammals, interferon-gamma-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. In this study a cDNA containing the orange-spotted grouper GILT (OsgGILT) coding sequence has been cloned and its complete sequence determined. The full-length cDNA of OsgGILT gene is 1066 bp nucleotides (nt) encoding a protein of 260 amino acids (aa), with a putative molecular weight of 28.

Construction of bifunctional fusion proteins consisting of duck BAFF and EGFP.

We constructed fusion proteins consisting of fluorescence-enhanced green fluorescent protein (EGFP) and soluble domain of duck B-cell-activating factor of the TNF family (dsBAFF). The soluble EGFP/dsBAFF was efficiently expressed in Escherichia coli BL 21 (DE3) and was purified in milligram amounts using metal chellate affinity chromatography. The fusion protein exhibited similar fluorescence spectra with free EGFP and promoted the survival of duck bursal B cells in vitro as well as dsBAFF.

cDNA cloning, expression and bioactivity of porcine BAFF.

B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF) is critical for B cell survival, maturation and T cell activation by acting through its three receptors, BAFF-R, BCMA and TACI. In the present study, a porcine BAFF cDNA, designated pBAFF, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pBAFF consists of 805bp with a 702bp open reading frame, encoding 233 amino acids.

Molecular cloning, in vitro expression and bioactivity of goose B-cell activating factor.

B-cell activating factor (BAFF), belonging to the TNF family, is critical for B cell survival and maturation. cDNA of goose BAFF (gBAFF) was amplified from goose spleen by RT-PCR. The open reading frame (ORF) of gBAFF encodes a protein of 288-amino acid.

Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.

The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid.

Cloning, expression and bioactivity of duck BAFF.

B cell activating factor (BAFF) belonging to the TNF family is critical for B cell survival and maturation. In the present study, we identified a duck BAFF cDNA, named dBAFF, by RT-PCR and RACE strategies. The open reading frame (ORF) of this cDNA encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like chicken BAFF (cBAFF), human BAFF (hBAFF) and mouse BAFF (mBAFF).