Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope

• Published in: Biotechnology letters 2006-10, Vol.28 (20), p.1649-1654
• Main Authors: Gao, Huiguang, Fu, Weiling, Li, Rongfen, Chen, Linfeng, Ji, Qing, Zhang, Li, Huang, Gang, He, Fengtian
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Antibody Formation; Autoimmune diseases; B lymphocyte stimulator; B-Cell Activating Factor - biosynthesis; B-Cell Activating Factor - genetics; B-Cell Activating Factor - immunology; B-Cell Activating Factor - isolation & purification; Bacterial proteins; Biological and medical sciences; Biotechnology; Cell Proliferation; Chromatography; E coli; Epitopes - genetics; Escherichia coli; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Immunization; Lymphocytes; Mice; Mice, Inbred BALB C; mutants; Mutation; Ovalbumin - genetics; Plasmids; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; T-helper cell epitope; T-Lymphocytes, Helper-Inducer - immunology
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-006-9139-y

The DNA encoding soluble B lymphocyte stimulator (134-285 amino acids, sBLyS) mutant with residues 217-224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5'-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5α after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.