title: Effect of N-linked glycosylation on secretion and activity of recombinant DSPAalpha1 expressed in Pichia pastoris.
abstract: The thrombolytic agent DSPAalpha1 is currently undergoing clinical trials for the treatment of acute ischemic stroke and has shown good pharmacodynamic, pharmacokinetic and safety profiles. Here, the DSPAalpha1 gene, optimized for the preferred codons of yeast, was cloned into the Pichia pastoris strains GS115 and KM71. Both expression systems produced functional DSPAalpha1 into the broth medium under shaking flask growth conditions with the yield of about 70 mg/L, and 105 mg/L, respectively. In addition, three glycosylation minus DSPAalpha1 mutants, constructed by site-directed mutagenesis, were also expressed in Pichia pastoris. The mutant proteins were assayed by SDS-PAGE and fibrin degradation activities were evaluated. The secretion levels of all the mutants, especially N362Q and N117Q/N362Q, were so lower compared to the wild-type DSPAalpha1 that only minimal quantities of mutant protein could be recovered by purification from the culture medium. The protein specific activities from mutants (N117Q, N362Q) were less 25% than that of the wild type protein. These results imply that the N-linked carbohydrate chains (at N117 and N362) are vital for the enzymatic activity of rDSPAalpha1 and for its secretion from Pichia pastoris.
authors.affiliation: The Research and Development Department, Qilu Pharmaceutical Co., Ltd., Jinan 250100, China.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Yan
authors.forename: Qingmin
authors.initials: Q
authors.lastname: Wang
authors.forename: Lixia
authors.initials: L
authors.lastname: Sun
authors.forename: Jingyi
authors.initials: J
authors.lastname: Wang
mesh_terms: D005343:Fibrinolytic Agents / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006031:Glycosylation; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010960:Plasminogen Activators / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D010960:Plasminogen Activators