Production and purification of recombinant human BLyS mutant from inclusion bodies.
B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor superfamily, is an important regulator of B cell homeostasis. In BLyS-deficient mice, B cell development is severely perturbed. On the other hand, mice transgenic for BLyS developed autoimmune disorders, such as increased germinal center formation, production of autoantibodies, and Ig deposition in kidneys. The overexpression of BLyS was found in some human autoimmune diseases. These findings suggest that BLyS has a crucial role in the humoral immune response and may be a therapeutic target for some human autoimmune diseases. To construct and express the therapeutic vaccine BLyS, we coupled a foreign immunodominant T-helper epitope to the N terminus of BLyS (named recombinant BLyS mutant, rBLySM) and expressed rBLySM in Escherichia coli. We have developed a purification process of rBLySM from inclusion bodies. A step-down urea concentration strategy was applied to the rBLySM renaturation process. By this strategy, a stable yield of 4.5mg purified rBLySM per gram of cell paste could be obtained.
Xue
Xiaochang
X
Biotechnology Center of The Fourth Military Medical University, Xi'an, PR China.
Wang
Zenglu
Z
Yan
Zhen
Z
Shi
Jihong
J
Han
Wei
W
Zhang
Yingqi
Y
D000595:Amino Acid Sequence; D053264:B-Cell Activating Factor; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013745:Tetanus Toxoid / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014614:Vaccines, Synthetic / Q000096:biosynthesis* / Q000302:isolation & purification
D053264:B-Cell Activating Factor; D018984:Epitopes, T-Lymphocyte; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C505719:TNFSF13B protein, human; D013745:Tetanus Toxoid; D014409:Tumor Necrosis Factor-alpha; D014614:Vaccines, Synthetic; D019791:Guanidine; D004229:Dithiothreitol