title: Simultaneous elimination of T- and B-cell epitope by structure-based mutagenesis of single Glu80 residue within recombinant staphylokinase.
abstract: To reduce the immunogenicity of recombinant staphylokinase, structure-based mutagenesis of Glu80 residue in wild-type staphylokinase (wt-Sak) was rationally designed and carried out by a modified QuikChange site-directed mutagenesis. Sak mutants, including Sak(E80A) and Sak(E80S), were successfully expressed in E. coli DH5a as a soluble cytoplasmic proteins and accounted for more than 40% of the total cellular proteins. The expressed proteins were purified by a three-step chromatographic purification process. SDS-PAGE and HPLC analyses results indicated that the purified proteins were almost completely homogeneous and the purities of Sak mutants exceeded 97%. Analysis of fibrinolytic activity revealed that substitution of E80 residue with serine and alanine resulted in slightly increased specific activities of Sak mutants. Investigation of the immunogenicity of Sak mutants showed that the amount of specific anti-Sak IgG antibodies elicited by Sak(E80A) and Sak(E80S) in BALB/c mice decreased approximately 35% and 27%, respectively compared with wt-Sak. The abilities of Sak mutants to stimulate proliferation of T cells from BALB/c mice and to bind mouse anti-Sak polyclonal serum were significantly lower than those of wt-Sak. These results suggested that substitution of Glu80 residue by alanine and serine successfully eliminated part of T- and B-cell epitope of Sak molecule. Our findings suggested that simultaneous elimination of T- and B-cell epitopes was a useful method to reduce the immunogenicity of wt-Sak molecule and provided a strategy for engineering safe Sak-based fibrinolytics for the clinical treatment of acute myocardial infarction.
authors.affiliation: College of Life Science, Hebei Normal University, Shijiazhuang 050016, China. he_jintian@yahoo.com
authors.forename: Jintian
authors.initials: J
authors.lastname: He
authors.forename: Ruiguang
authors.initials: R
authors.lastname: Xu
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Kai
authors.initials: K
authors.lastname: Jia
authors.forename: Xianglian
authors.initials: X
authors.lastname: Zhou
authors.forename: Ke
authors.initials: K
authors.lastname: Zhu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018985:Epitopes, B-Lymphocyte / Q000235:genetics* / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000235:genetics* / Q000276:immunology*; D005260:Female; D018698:Glutamic Acid / Q000235:genetics; D008666:Metalloendopeptidases / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D011994:Recombinant Proteins; D018698:Glutamic Acid; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus