Construction and characterization of a bi-functional EGFP/sBAFF fusion protein.
A fusion between gene encoding fluoresce-enhanced green fluorescent protein variant (EGFP) and soluble domain of human B-cell-activating factor of the TNF family (sBAFF) was constructed and expressed in Escherichia coli. The EGFP/sBAFF had an apparent molecular weight of 45 kDa and was detected with anti-hsBAFF and anti-His(6) monoclonal antibodies. After being purified by immobilized metal affinity chromatography (IMAC), the fusion protein retained similar fluorescence spectra to those of EGFP. Biological activity assays showed the EGFP/sBAFF as well as sBAFF could co-stimulated human B lymphocyte proliferation in vitro. In addition, EGFP/sBAFF has shown specific binding to BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active sBAFF that may prove useful in further studies on BAFF and its receptors.
Cao
Peng
P
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210097, Jiangsu, China.
Zhang
Shuangquan
S
Zhang
Juan
J
Wang
Min
M
D053264:B-Cell Activating Factor / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D053264:B-Cell Activating Factor; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins