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The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in promoting the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens. It is also involved in MHC I-restricted antigens catalyzing disulfide bond reduction in fishes' adaptive immunity. The open reading frame of tongue sole (Cynoglossus semilaevis) GILT (tsGILT) gene is 771 bp long, encoding 257 amino acids, with a calculated molecular weight of 28.465 kDa and isoelectric point (pI) of 5.35. After induction with lipopolysaccharide, the expression of tsGILT mRNA was upregulated in spleen and kidney and recombinant tsGILT protein transferred to late endosomes and lysosomes in HeLa cells. The refolded tsGILT was capable of catalyzing the reduction of the interchain disulfide bonds against an IgG substrate depend on the active site CXXC motif at residues 75–78. The process of immune response to bacteria challenge needs GILT to catalyze the reduction of disulfide bond and unfolding native protein antigens, promoting their hydrolysis by proteases. Whether a single mutation or a double mutation of active site CXXC at residues75–78, the 3D structure of tsGILT protein has undergone major changes and lost its activity of catalyzing the reduction of the interchain disulfide bonds. • tsGILT is constitutively expressed in a tissue-specific manner. • tsGILT expression is up-regulated in spleen and kidney cells following LPS stimulation. • tsGILT translocated to late endosomes and lysosomes under the condition of LPS stimulation. • tsGILT exhibited thiol reductase activity in vitro. • Mutation of one or double cysteine residues of CXXC active site abolished thiol reductase activity. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, the cDNA encoding the extracellular soluble domain of rabbit BAFF (rsBAFF) was subcloned into the pET28a vector and the recombinant rsBAFF protein was solubly produced in Escherichia coli. Recombinant rsBAFF protein of 17.1kDa was then purified to a purity of 95% by using Ni2+-IDA resin. In vitro, purified rsBAFF protein was able to promote the survival/proliferation of B-lymphocytes of rabbits, mice and humans in the presence of anti-IgM antibodies. These findings indicate that the recombinant rsBAFF produced in E. coli have the bioactivity, and functional cross-reactivity occurs between rabbit and other mammalians BAFF proteins. [Copyright &y& Elsevier]