Cloning, expression and bioactivity of BAFF from Petaurus breviceps.
B-cell activating factor (BAFF), belonging to the TNF (tumor necrosis factor) family, is crucial for B-cell survival and maturation. In the present study, PbBAFF cDNA was amplified from the sugar glider Petaurus breviceps by RT-PCR and RACE (rapid amplification of cDNA ends) strategies. The open reading frame (ORF) of PbBAFF cDNA encodes a protein consisting of 287-amino acid. The deduced amino acid sequence contains a predicted transmembrane domain, a putative furin protease cleavage site, a potential N-glycosylation site and conserved cysteine residues similar to that identified in other mammalian BAFF. The soluble mature part of PbBAFF (PbsBAFF) showed 88-92% sequence identity with mammalian homologs. The predicted three-dimensional (3D) structural analysis of PbsBAFF analyzed by comparative protein modeling revealed that they are very similar to the 3D structure of human BAFF. Recombinant PbsBAFF fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, purified PbsBAFF co-stimulates the proliferation of human B-cells. These findings indicate PbBAFF, the first BAFF cloned from marsupial, plays an important role in proliferation of B-cells, and phylogenetic analyses reveal that the work is of value with respect to continued refinement of our understanding of mammalian phylogenetic relationships.
Ren
Wen-hua
WH
Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.
Xu
Ying
Y
Zhang
Shuang-quan
SQ
Yang
Guang
G
D000595:Amino Acid Sequence; D000818:Animals; D053264:B-Cell Activating Factor / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001402:B-Lymphocytes / Q000276:immunology; D003001:Cloning, Molecular; D006801:Humans; D008213:Lymphocyte Activation; D008394:Marsupialia / Q000276:immunology*; D008969:Molecular Sequence Data; D010802:Phylogeny
D053264:B-Cell Activating Factor