The construction and characterization of a bifunctional EGFP/sAPRIL fusion protein.
A fusion protein of enhanced green fluorescent protein (EGFP) and soluble domain of human a proliferation-inducing ligand (sAPRIL) was efficiently expressed in Escherichia coli BL 21 (DE3). The soluble EGFP/sAPRIL, around 43 kDa, was purified in milligram amounts using metal chellate affinity chromatography and detected with anti-His(6) and anti-hsAPRIL monoclonal antibody. The chimeric protein exhibited similar fluorescence spectra with free EGFP. In vitro, purified EGFP/sAPRIL specifically bound receptor B cell maturation antigen (BCMA) detected by enzyme linked immunosorbent assay (ELISA) and receptors [including heparan sulfate proteoglycan (HSPGs)]-positive cell lines analyzed by fluorescence-activated cell sorting (FACS). Confocal laser microscopy images visibly showed the HSPGs'-dependent binding of EGFP/sAPRIL to NIH-3T3 cell. In addition, the chimera retained the bioactivity to stimulate/co-stimulate proliferation of NIH-3T3 and Jurkat cell/human B cell in vitro. Therefore, the fusion protein shows a readily obtainable source of biologically active sAPRIL which has considerable potential for single-step fluorescence detection assay in the study of APRIL and its receptors.
Guan
Zhengbing
Z
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210097, China.
Yao
Wenjuan
W
Ye
Jilin
J
Dan
Wenbing
W
Shen
Jiayin
J
Zhang
Shuangquan
S
D000818:Animals; D053301:B-Cell Maturation Antigen / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear; D008213:Lymphocyte Activation; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13 / Q000235:genetics / Q000378:metabolism*
D053301:B-Cell Maturation Antigen; D019812:Heparan Sulfate Proteoglycans; D011993:Recombinant Fusion Proteins; C086966:TNFRSF17 protein, human; C114631:TNFSF13 protein, human; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins